Nucleic acid labeling compounds

ABSTRACT

Nucleic acid labeling compounds containing heterocyclic derivatives are disclosed. Methods for making such heterocyclic compounds are also disclosed. The labeling compounds are suitable for enzymatic attachment to a nucleic acid, either terminally or internally, to provide a mechanism of nucleic acid detection.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/641,677, filed Aug. 15, 2003, which application is acontinuation-in-part of U.S. application Ser. No. 10/314,012, filed Dec.5, 2002, which is a continuation-in-part of U.S. application Ser. No.10/097,113, filed Mar. 12, 2002; and a continuation-in-part of U.S.application Ser. No. 09/952/387, filed Sep. 11, 2001, acontinuation-in-part of U.S. application Ser. No. 09/780,574, filed Feb.9, 2001, continuation-in-part of U.S. application Ser. No. 09/126,645,filed Jul. 31, 1998, and a continuation-in-part of U.S. Ser. No.08/882,649, filed: Jun. 25, 1997 which is a continuation ofPCT/US97/01603, filed on Jan. 22, 1997 designating the Unites States ofAmerica, which claims priority to U.S. Provisional Application No.60/010,471 filed on Jan. 23, 1996 and U.S. Provisional Application No.60/035,170, filed on Jan. 9, 1997, all of which are herein incorporatedby reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This invention was made with Government support under contract70NANB5H1031 awarded by the Advanced Technology Program of the NationalInstitute of Standards and Technology. The Government has certain rightsin this invention.

BACKGROUND OF THE INVENTION

Gene expression in diseased and healthy individuals is oftentimesdifferent and characterizable. The ability to monitor gene expression insuch cases provides medical professionals with a powerful diagnostictool. This form of diagnosis is especially important in the area ofoncology, where it is thought that the overexpression of an oncogene, orthe underexpression of a tumor suppressor gene, results intumorogenesis. See Mikkelson et al. J. Cell. Biochem. 1991, 46, 3-8.

One can indirectly monitor gene expression, for example, by measuringnucleic acid (e.g., mRNA) that is the transcription product of atargeted gene. The nucleic acid is chemically or biochemically labeledwith a detectable moiety and allowed to hybridize with a localizednucleic acid of known sequence sometimes, know here as a probe. Thedetection of a labeled nucleic acid at the probe position indicates thatthe targeted gene has been expressed. See International ApplicationPublication Nos. WO 97/27317, WO 92/10588 and WO 97/10365.

The labeling of a nucleic acid is typically performed by covalentlyattaching a detectable group (label) to either an internal or terminalposition. Scientists have reported a number of detectable nucleotideanalogues that have been enzymatically incorporated into an oligo- orpolynucleotide. Langer et al., for example, disclosed analogues of dUTPand UTP that contain a covalently bound biotin moiety. Proc. Natl. Acad.Sci. USA 1981, 78, 6633-6637. The analogues, shown below, possess anallylamine linker arm that is attached to the C-5 position of thepyrimidine ring at one end and a biotin moiety at the other. The dUTPand UTP analogues, wherein R is H or OH, were incorporated into apolynucleotide.

Petrie et al. disclosed a dATP analogue,3-[5-[(N-biotinyl-6-aminocaproyl)-amino]pentyl]-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine-5′-triphosphate.Bioconjugate Chem. 1991, 2, 441-446. The analogue, shown below, ismodified at the 3-position with a linker arm that is attached to abiotin moiety. Petrie et al. reported that the compound wherein R isbiotin is incorporated into DNA by nick translation.

Prober et al. disclosed a set of four dideoxynucleotides, eachcontaining a succinylfluorescein dye. Science 1987, 238, 336-341. Thedideoxynucleotides, one of which is shown below, were enzymaticallyincorporated into an oligonucleotide through a template directedextension of a primer. The compounds provided for a DNA sequencingmethod based on gel migration.

Herrlein et al. disclosed modified nucleoside trisphosphates of the fourDNA bases. Helv. Chim. Acta 1994, 77, 586-596. The compounds, one ofwhich is shown below, contain a 3′-amino group containing radioactive orfluorescent moieties. Herrlein et al. further described the use of thenucleoside analogues as DNA chain terminators.

Cech et al. disclosed 3′-amino-functionalized nucleoside triphosphates.Collect. Czech. Chem. Commun. 1996, 61, S297-S300. The compounds, one ofwhich is shown below, contain a fluorescein attached to the 3′-positionthrough an amino linker. Cech et al. proposed that the describedfunctionalized nucleosides would be useful as terminators for DNAsequencing.

SUMMARY OF THE INVENTION

The present invention relates to nucleic acid labeling compounds. Morespecifically, the invention provides heterocyclic derivatives containinga detectable moiety. It further provides methods of attaching theheterocyclic derivatives to a nucleic acid.

The present invention provides nucleic acid labeling compounds that arecapable of being enzymatically incorporated into a nucleic acid. Thenucleic acids to which the compounds are attached substantially maintaintheir ability to bind to a complementary nucleic acid sequence.

DISCLOSURE OF THE INVENTION

The present invention relates to nucleic acid labeling compounds. Morespecifically, the invention provides heterocyclic derivatives containinga detectable moiety. The invention also provides methods of making suchheterocyclic derivatives. It further provides methods of attaching theheterocyclic derivatives to a nucleic acid.

The development of a novel nucleic acid labeling compound that iseffectively incorporated into a nucleic acid to provide a readilydetectable composition would benefit genetic analysis technologies. Itwould aid, for example, in the monitoring of gene expression and thedetection and screening of mutations and polymorphisms. Such a compoundshould be suitable for incorporation into a nucleic acid either byenzymatic or other means. Furthermore, the nucleic acid to which thelabeling compound is attached should maintain its ability to bind to aprobe, such as a complementary nucleic acid.

The present invention provides nucleic acid labeling compounds that arecapable of being enzymatically incorporated into a nucleic acid. Thenucleic acids to which the compounds are attached substantially maintaintheir ability to bind to a complementary nucleic acid sequence.

One aspect of the instantly disclosed invention are nucleic acidlabeling compounds of the following structure:A-O—CH₂-T-H_(c)-L-(M)_(m)-Qwherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; T is a templatemoiety; He is a heterocyclic group, includuding without limitation abase such as A, T, G, C, and U; L is a linker moiety; Q is a detectablemoiety; and M is a connecting group, wherein m is an integer rangingfrom 0 to about 5. Thus, the connecting group may optionally not bepresent, depending on, inter alia the nature of L and Q.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

The invention also contemplates steroisomers two of which are shownbelow by way of example:

In the above molecules, A is H or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid byeither enzymatic or, e.g., by chemical means;

-   -   X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H,        alkyl or aryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H,        alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or        aryl; L is a linker group, preferably an amido alkyl; Q is a        detectable moiety; and, M is a connecting group, wherein m is an        integer ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 2 to about 10; Q is biotin or a carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

In another embodiment, Y is H or OH; Z is H or OH; L is—C(O)NH(CH₂)₄NH—; Q is biotin; and, M is —CO(CH₂)₅NH, wherein m is 1.

In another embodiment, Y is H or OH; Z is H or OH; L is—C(O)NH(CH₂)₄NH—; Q is 5-carboxyfluorescein and, m is 0.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

The present invention includes stereoisomers of the above compound, twoof which are shown below by example.

With respect to these disclosed compounds, A is H or a functional groupthat permits the attachment of the nucleic acid labeling compound to anucleic acid; X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are,independently, H, alkyl or aryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉,wherein R₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H,alkyl or aryl; L is a linking group, preferably amino alkyl; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 2 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH₂)₄NH—; Qis biotin; and, m is 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH₂)₄NH—; Qis 5-carboxyfluorescein; and, m is 0.

In one embodiment, the nucleic acid labeling compounds have thefollowing structure:

The present invention includes stereoisomers, two examples of which areshown below:

In these compounds, A is H or a functional group that permits theattachment of the nucleic acid labeling compound to a nucleic acid; X isO, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H, alkyl oraryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Zis H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is a linkermoiety, preferably alkynyl alkyl; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —C≡C(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH—, wherein m is 1 or 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —C≡CCH₂NH—; Q isbiotin; and, m is 1.

In another embodiment, Y is H or OH; Z is H or OH; L is —C≡CCH₂NH—; Q is5-carboxyfluorescein; and, m is 1.

In another embodiment L is selected from the group consisting of—CH═CH—C(O)— and —CH═CH—(CH₂)_(n)NH— where n is an interger from 1 toabout 5

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group, preferablyan amino alkyl; Q is a detectable moiety; and, M is a connecting group,wherein m is an integer ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —NH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 2 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH₂)₄NH—; Qis biotin; and, M is —CO(CH₂)₅NH—, wherein m is 1.

In another embodiment, Y is H or OH; Z is H or OH; L is —NH(CH₂)₄NH—; Qis 5-carboxyfluorescein; and, M is —CO(CH₂)₅NH—, wherein m is 1.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H.N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is a linker group,preferably a functionalized alkyl, alkenyl alkyl or alkynyl alkyl; Q isa detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or carboxyfluorescein; and, M is—CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CHCH₂NH—; Qis biotin; and, m is 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CHCH₂NH—; Qis 5-carboxyfluorescein; and, m is 0.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is a linker group,preferably a functionalized alkyl, alkenyl alkyl or alkynyl alkyl; Q isa detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is 0; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is a linker group, preferably —CH═CH(CH₂)_(n)NH—, wherein nis an integer ranging from about 1 to about 10; Q is biotin orcarboxyfluorescein; and, M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—,wherein m is 1.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CHCH₂NH—; Qis biotin; and, m is 0.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CHCH₂NH—; Qis 5-carboxyfluorescein; and, m is 0.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group, preferablyfunctionalized alkyl; Q is a detectable moiety; and, M is a connectinggroup, wherein m is an integer ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is 0; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or fluorescein; and, M is—NH(CH₂CH₂O)_(k)NH— or, more preferably, —NH(CH₂CH₂O)_(k)CH₂CH₂NH—,wherein, k is an integer from 1 to about 5, wherein m is 1 and k ispreferably 1.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH₂—C(O)—; Q isa carboxyfluorescein or biotin; and M is —NH(CH₂CH₂O)_(k)NH— or, morepreferably NH(CH₂CH₂O)_(k)CH₂CH₂NH—, wherein, k is 2 and m is 1.

In another embodiment, Y is OH; Z is OH; L is —CH₂—C(O)—; Q is biotin;and M is —NH(CH₂CH₂O)_(k)NH— or more preferably—NH(CH₂CH₂O)_(k)CH₂CH₂NH—, wherein, k is 2 and m is 1.

In another embodiment,; L is —CH═CHCH₂NH—; Q is a carboxyfluorescein;and M is —NH(CH₂CH₂O)_(k)NH— or more preferably—NH(CH₂CH₂O)_(k)CH₂CH₂NH—, wherein, k is 2 and m is 1.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or a fluorescein; and, a first M is—NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —(CH₂)₂C(O)—, Qis biotin or a carboxyfluorescein; and a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is acarboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and a second M is—CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is biotin;and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, whereinm is 2.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid,preferably A is a triphosphate group with apporpriate counterions. Thecounterions are selected from the group consisting of H+, Na+, Li+, K+,and NH₄+; X is O; Y is OH; Z is OH; L is selected from the groupconsisting of —CH═CH—C(O)—NH—CH₂—CH₂—NH—C(O)— and—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O); M is —(CH₂)₅—NH— and Q is biotinhaving the structure:

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is selected from the group consisting of —CH═CH—C(O)— and—CH═CH—CH(NH₂)—; Q is biotin or a fluorescein; and, a first M is—NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10, and asecond M is —CO(CH₂)_(p)NH—, wherein p is an interger from about two toabout 10 and m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CH—C(O)—, Qis biotin or a carboxyfluorescein; and a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —CH═CH—C(O)—, Q is abiotin or a carboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —CH═CH—C(O)—, Q is orbiotin; and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—,wherein m is 2.

In yet another embodiment L comprises a vinyl group (—CH═CH—) attachedat its 1 position directly to the N of the base. In accordance with thisaspect of the present invention, where L is vinyl, further substituentsmay be attached to the 2 position of the vinyl groups such as such asfor example R₁₁ (—CH═CH—R₁₁). Preferably R₁₁ is alkyl, aryl,functionalized alkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and aminoalkyl. Most preferablbly R₁₁ is C(O)R₁₂ where R₁₂ is a bond, aryl,functionalized alkyl, amido alkyl, alkenyl alkyl alkoxy, thio and aminoalkyl.

Importantly, as disclosed in accordance with one aspect of the presentinvention, the linker group L is selected to provide a linking function,which either alone or in conjunction with appropriate connecting group(M), provide appropriate spacing of the Q group from the Hc or basegroup at such a length and in such a configuration as to allow anappropriate assay to be performed on the Q group, but at the same timesubstantially preserving the ability of the nucleic acid labelingcompound to act as a substrate for the appropriate enzyme, e.g.,terminal transferase and/or RNA polymerase. Those of skill in the artwill also appreciate that the Hc-L-(M)m-Q groups must be chosen, inaccordance with the present invention, to avoid subtantially inhibitingthe ability of a nucleic acid strand incorporating such group to undergoWatson-Crick type base pairing with complementary sequences. Thus,-L-(M)m-Q may be any arrangements or grouping of molecules or atomswhich functions to allow nucleic acids to be labeled and detected.

In accordance with one aspect of the present invention, R₁₂ ispreferably a bond, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, and Q isselected from the group consisting of a fluorescein and a biotin; and afirst M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, wherein m is 2.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is functionalized alkyl; Qis a detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; ; L is —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or a fluorescein; and, a first M is—NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —(CH₂)₂C(O)—, Qis biotin or a carboxyfluorescein; and a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is acarboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and a second M is—CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is biotin;and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, whereinm is 2.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+; X is O; Y is OH; Z is OH; L is selected from the group consistingof —CH═CH— C(O)—NH—CH₂—CH₂—NH—C(O)— and—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O); M is —(CH₂)₅—NH— and Q is biotinhaving the structure:

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —CH═CH—C(O)—; Q is biotin or a fluorescein; and, a first Mis —NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10,and a second M is —CO(CH₂)_(p)NH—, wherein p is an interger from about 2to about 10 and m is 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —CH═CH—C(O)—, Qis biotin or a carboxyfluorescein; and a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —CH═CH—C(O)—, Q is abiotin or a carboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —CH═CH—C(O)—, Q is biotin;and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, whereinm is 2.

In another embodiment L comprises a vinyl group (—CH═CH—) attached atits 1 position directly to the N of the base. In accordance with thisaspect of the present invention, where L is vinyl, further substituentsare attached to the 2 position of the vinyl groups such as such as forexample R₁₁ (—CH═CH—R₁₁). Preferably R₁₁ is alkyl, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Mostpreferablbly R₁₁ is C(O)R₁₂, where R₁₂ is a bond, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.

Importantly, as disclosed in accordance with one aspect of the presentinvention, the linker group L is selected to provide a linker function,which either alone or in conjunction with appropriate connecting groups(M), appropriately spaces the Q group from the Hc or base group at sucha length and in such a configuration as to allow an appropriate assay tobe performed on the Q group, but at the same time substantiallypreservers the ability of the nucleic acid labeling compound to act as asubstrate for the appropriate enzyme, e.g., terminal transferase and/orRNA polymerase. Those of skill in the art will also appreciate that thechosen Hc-L-(M)m-Q groups must be chosen in accordance with the presentinvention, to avoid subtantially inhibiting the ability of a nucleicacid strand incorporating such group to undergo Watson-Crick type basepairing with complementary sequences. Thus, -L-(M)m-Q may be anyarrangements or gourping of molecules or atoms which functions to allownucleic acids to be labeled and detected.

In accordance with one aspect of the present invention, R₁₂ ispreferably a bond, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, and Q isselected from the group consisting of a fluorescein and a biotin; and afirst M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, wherein m is 2.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is a linking group,preferably amido alkyl; Q is a detectable moiety; and, M is a connectinggroup, wherein m is an integer ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging fromabout 2 to about 10; Q is biotin or a fluorescein; wherein m is 0, 1, or2.

In another embodiment, Y is H or OH; Z is H or OH; L is —C(O)NH(CH₂)₄NH—or, more preferably, —C(O)NH(CH₂)₂NH—; M is preferably —C(O)(CH₂)₅NH andQ is biotin or a carboxyfluorescein.

In another embodiment, Y is OH; Z is H; L is —C(O)NH(CH₂)₄NH— or, morepreferably, —C(O)NH(CH₂)₂NH—; M is preferably —C(O)(CH₂)₅NH; Q isbiotin.

In another embodiment, Y is OH; Z is H; L is —C(O)NH(CH₂)₄NH— or, morepreferably, —C(O)NH(CH₂)₂NH—; M is preferably —C(O)(CH₂)₅NH; and Q is acarboxyfluorescein.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+, Y is OH, X is OH, Z is H or OH, L is —C(O)NH(CH₂)₂NH—, M is—C(O)(CH₂)₅NH—, n is 1 and Q is biotin, having the structure:

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+, Y is OH, X is O, Z is H or OH, L is —C(O)NH(CH₂)₂NH—, M is—C(O)((CH₂)₂O)₄(CH₂)₂NH—, n is 1 and Q is biotin, having the structure:

In another aspect of the present invention, a method for preparing alabeled nucleic acid sample is provided having the steps of: providing anucleic acid sample, the nucleic acid sample comprising DNA; reactingthe nucleic acid sample in the presence of the enzyme terminaltransferase with the preceding nucleic acid labeling compound.Preferably, according to the instant invention that nucleic acid sampleis cDNA.

The present invention also provides nucleic acid derivatives produced bycoupling a nucleic acid labeling compound with a nucleic acid andhybridization products comprising the nucleic acid derivatives bound toa complementary probe.

In one embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or a carboxyfluorescein; and, M is —CO(CH₂)₅NH—,wherein m is 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—C≡C(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH—, wherein mis 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is functionalized alkyl; Qis a detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

In another embodiment, the nucleic acid labeling compounds used in thecoupling have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

The hybridization product formed from this nucleic acid derivativecomprises the nucleic acid derivative bound to a complementary probe. Inone embodiment, the probe is attached to a glass chip.

The present invention also provides methods of synthesizing nucleic acidderivatives by attaching a nucleic acid labeling compound to a nucleicacid. It further provides methods of detecting nucleic acids involvingincubating the nucleic acid derivatives with a probe.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—C(O)NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 toabout 10; Q is biotin or a carboxyfluorescein; and, M is —CO(CH₂)₅NH—,wherein m is 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—C≡C(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH—, wherein mis 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or H₄O₉P₃—; X is O; Y is H or OR₉, wherein R₉ is H, alkylor aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from about 1 toabout 10; Q is biotin or carboxyfluorescein; and, M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl, aryl or afunctionalized alkyl or aryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; L is linker group; Q is a detectable moiety; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is functionalized alkyl; Qis a detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In one embodiment, the nucleic acid labeling compounds attached to thenucleic acid have the following structures for example:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is linker group; Q is adetectable moiety; and, M is a connecting group, wherein m is an integerranging from 0 to about 3.

The method of nucleic acid detection using the nucleic acid derivativeinvolves the incubation of the derivative with a probe. In oneembodiment, the probe is attached to a glass chip.

In yet another embodiment, the methods involve the steps of: (a)providing at least one nucleic acid coupled to a support; (b) providinga labeled moiety capable of being coupled with a terminal transferase tosaid nucleic acid; (c) providing said terminal transferase; and (d)coupling said labeled moiety to said nucleic acid using said terminaltransferase.

In still another embodiment, the methods involve the steps of: (a)providing at least two nucleic acids coupled to a support; (b)increasing the number of monomer units of said nucleic acids to form acommon nucleic acid tail on said at least two nucleic acids; (c)providing a labeled moiety capable of recognizing said common nucleicacid tails; and (d) contacting said common nucleic acid tails and saidlabeled moiety.

In still yet another embodiment, the methods involve the steps of: (a)providing at least one nucleic acid coupled to a support; (b) providinga labeled moiety capable of being coupled with a ligase to said nucleicacid; (c) providing said ligase; and (d) coupling said labeled moiety tosaid nucleic acid using said ligase.

This invention also provides compounds of the formulas described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a nonlimiting set of template moieties.

FIG. 2 shows a nonlimiting set of heterocyclic groups:4-aminopyrazolo[3,4-d]pyrimidine, pyrazolo[3,4-d]pyrimidine, 1,3-diazole(imidazole), 1,2,4-triazine-3-one, 1,2,4-triazine-3,5-dione and5-amino-1,2,4-triazine-3-one.

FIG. 3 shows a synthetic route to fluorescein and biotin labeled1-(2,3-dideoxy-D-glycero-pentafuranosyl)imidazole-4-carboxamidenucleotides.

FIG. 4 shows a synthetic route to C3-labeled4-aminopyrazolo[3,4-d]pyrimidine β-D-ribofuranoside triphosphates.

FIG. 5 shows a synthetic route to fluorescein and biotin labeledN6-dideoxy-pyrazolo[3,4-d]pyrimidine nucleotides.

FIG. 6 shows a synthetic route to N4-labeled 1,2,4-triazine-3-oneβ-D-ribofuranoside triphosphates.

FIG. 7 shows a synthetic route to biotin and fluorescein C5-labeled1,2,4-triazine-3,5-dione riboside triphosphates.

FIG. 8 shows a synthetic route to biotin and fluorescein C5-labeled5-amino-1,2,4-triazine-3-one riboside triphosphates.

FIG. 9 shows graphical comparisons of observed hybridizationfluorescence intensities using Fluorescein-ddITP and Fluorescein-ddATP.

FIG. 10 shows a graphical comparison of observed hybridizationfluorescence intensities using Biotin-(M)₂-ddAPTP (whereinM=aminocaproyl) and Biotin-N6-ddATP.

FIG. 11 shows graphical comparisons of observed hybridizationfluorescence intensities using Biotin-M-ddITP (wherein M=aminocaproyl)and Biotin-N6-ddATP.

FIG. 12 shows a graphical comparison of overall re-sequencing(base-calling) accuracy using Fluorescein-ddITP and Fluorescein-N6-ddATPlabeled targets.

FIG. 13 shows a graphical comparison of overall re-sequencing accuracyusing Biotin-M-ddITP (wherein M=aminocaproyl) and Biotin-N6-ddATP.

FIG. 14 shows a graphical comparison of re-sequencing accuracy usingBiotin-(M)₂-ddAPPTP (wherein M=aminocaproyl) and Biotin-N6-ddATP.

FIG. 15 shows a schematic for the preparation of N1-labeled3-(β-D-ribofuranosyl)-1H-pyrazalo-[4,3-d]pyrimidine 5′-triphosphate.

FIG. 16 shows a schematic for the preparation of N1-labeled5-(β-D-ribofuranosyl)-2,4[1H,3H]-pyrimidinedione 5′-triphosphate.

FIG. 17 shows a schematic for the preparation of N-labeled2,5-anhydro-3-deoxy-D-ribo-hexamide 6-triphosphate.

FIG. 18 shows various labeling reagents suitable for use in the methodsdisclosed herein. FIG. 18 a shows various labeling reagents. FIG. 18 bshows still other labeling reagents. FIG. 18 c shows non-ribose ornon-2′-deoxyribose-containing labels. FIG. 18 d shows sugar-modifiednucleotide analogue labels 18 d.

FIG. 19 shows HIV array data for analog 42a (T7 labeling of RNA target).

FIG. 20 shows HPLC incorporation efficiency of C-nucleotide 42a (T7 RNApol, 1 kb transcript).

FIG. 21 shows IVT incorporation of saturated versus unsaturated nucleicacid labeling compounds.

DEFINITIONS

“Alkyl” refers to a straight chain, branched or cyclic chemical groupcontaining only carbon and hydrogen. Alkyl groups include, withoutlimitation, ethyl, propyl, butyl, pentyl, cyclopentyl and 2-methylbutyl.Alkyl groups are unsubstituted or substituted with 1 or moresubstituents (e.g., halogen, alkoxy, amino).

“Aryl” refers to a monovalent, unsaturated aromatic carbocyclic group.Aryl groups include, without limitation, phenyl, naphthyl, anthryl andbiphenyl. Aryl groups are unsubstituted or substituted with 1 or moresubstituents (e.g. halogen, alkoxy, amino).

“Amido alkyl” refers to a chemical group having the structure—C(O)NR₃R₄—, wherein R₃ is hydrogen, alkyl or aryl, and R₄ is alkyl oraryl. Preferably, the amido alkyl group is of the structure—C(O)NH(CH₂)_(n)R₅—, wherein n is an integer ranging from about 2 toabout 10, and R₅ is O, NR₆, or C(O), and wherein R₆ is hydrogen, alkylor aryl. More preferably, the amido alkyl group is of the structure—C(O)NH(CH₂)_(n)N(H)—, wherein n is an integer ranging from about 2 toabout 6. Most preferably, the amido alkyl group is of the structure—C(O)NH(CH₂)₄N(H)—.

“Alkynyl alkyl” refers to a chemical group having the structure—C≡C—R₄—, wherein R₄ is alkyl or aryl. Preferably, the alkynyl alkylgroup is of the structure —C≡C—(CH₂)_(n)R₅—, wherein n is an integerranging from 1 to about 10, and R₅ is O, NR₆ or C(O), wherein R₆ ishydrogen, alkyl or aryl. More preferably, the alkynyl alkyl group is ofthe structure —C═C—(CH₂)_(n)N(H)—, wherein n is an integer ranging from1 to about 4. Most preferably, the alkynyl alkyl group is of thestructure —C≡C—CH₂N(H)—.

“Alkenyl alkyl” refers to a chemical group having the structure—CH═CH—R₄—, wherein R₄ is alkyl or aryl. Preferably, the alkenyl alkylgroup is of the structure —CH═CH—(CH₂)_(n)R₅—, wherein n is an integerranging from 1 to about 10, and R₅ is O, NR₆ or C(O), wherein R₆ ishydrogen, alkyl or aryl. More preferably, the alkenyl alkyl group is ofthe structure —CH═CH—(CH₂)_(n)N(H)—, wherein n is an integer rangingfrom 1 to about 4. Most preferably, the alkenyl alkyl group is of thestructure —CH═CH—CH₂N(H)—.

“Functionalized alkyl” refers to a chemical group of the structure—(CH₂)_(n)R₇—, wherein n is an integer ranging from 1 to about 10, andR₇ is O, S, NH or C(O). Preferably, the functionalized alkyl group is ofthe structure —(CH₂)_(n)C(O)—, wherein n is an integer ranging from 1 toabout 4. More preferably, the functionalized alkyl group is of thestructure —CH₂C(O)—.

“Alkoxy” refers to a chemical group of the structure —O(CH₂)_(n)R₈—,wherein n is an integer ranging from 2 to about 10, and R₈ is O, S, NHor C(O). Preferably, the alkoxy group is of the structure—O(CH₂)_(n)C(O)—, wherein n is an integer ranging from 2 to about 4.More preferably, the alkoxy group is of the structure —OCH₂CH₂C(O)—.

“Thio” refers to a chemical group of the structure —S(CH₂)_(n)R₈—,wherein n is an integer ranging from 2 to about 10, and R₈ is O, S, NHor C(O). Preferably, the thio group is of the structure—S(CH₂)_(n)C(O)—, wherein n is an integer ranging from 2 to about 4.More preferably, the thio group is of the structure —SCH₂CH₂C(O)—.

“Amino alkyl” refers to a chemical group having an amino group attachedto an alkyl group. Preferably an amino alkyl is of the structure“NH(CH₂)_(n)NH—, wherein n is an integer ranging from about 2 to about10. More preferably it is of the structure —NH(CH₂)_(n)NH—, wherein n isan integer ranging from about 2 to about 4. Most preferably, the aminoalkyl group is of the structure —NH(CH₂)₄NH—.

“Nucleic acid” refers to a polymer comprising 2 or more nucleotides andincludes single-, double- and triple stranded polymers. “Nucleotede”refers to both naturally occurring and non-naturally occurring compoundsand comprises a heterocyclic base, a sugar, and a linging group,preferably a phosphate ester. For example, structural groups may beadded to the ribosyl or deoxyribosyl unit of the nucleotide, such as amethyl or allyl group at the 2′-O position or a fluoro group thatsubstitutes for the 2′-O group. The linking group, such as aphosphodiester, of the nucleic acid may be substituted or modified, forexample with methyl phosphonates or O-methyl phosphates. Bases andsugars can also be modified, as is known in the art. “Nucleic acid,” forthe purposes of this disclosure, also includes “peptide nucleic acids”in which native or modified nucleic acid bases are attached to apolyamide backbone.

The phrase “coupled to a support” means bound directly or indirectlythereto including attachment by covalent binding, hydrogen bonding,ionic interaction, hydrophobic interaction, or otherwise.

“Probe” refers to a nucleic acid that can be used to detect, byhybridization, a target nucleic acid. Preferably, the probe iscomplementary to the target nucleic acid along the entire length of theprobe, but hybridization can occur in the presence of one or more basemismatches between probe and target.

“Perfect match probe” refers to a probe that has a sequence that isperfectly complementary to a particular target sequence. The test probeis typically perfectly complementary to a portion (subsequence) of thetarget sequence. The perfect match (PM) probe can be a “test probe”, a“normalization control” probe, an expression level control probe and thelike. A perfect match control or perfect match probe is, however,distinguished from a “mismatch control” or “mismatch probe.” In the caseof expression monitoring arrays, perfect match probes are typicallypreselected (designed) to be complementary to particular sequences orsubsequences of target nucleic acids (e.g., particular genes). Incontrast, in generic difference screening arrays, the particular targetsequences are typically unknown. In the latter case, prefect matchprobes cannot be preselected. The term perfect match probe in thiscontext is to distinguish that probe from a corresponding “mismatchcontrol” that differs from the perfect match in one or more particularpreselected nucleotides as described below.

“Mismatch control” or “mismatch probe”, in expression monitoring arrays,refers to probes whose sequence is deliberately selected not to beperfectly complementary to a particular target sequence. For eachmismatch (MM) control in a high-density array there preferably exists acorresponding perfect match (PM) probe that is perfectly complementaryto the same particular target sequence. In “generic” (e.g., random,arbitrary, haphazard, etc.) arrays, since the target nucleic acid(s) areunknown perfect match and mismatch probes cannot be a priori determined,designed, or selected. In this instance, the probes are preferablyprovided as pairs where each pair of probes differ in one or morepreselected nucleotides. Thus, while it is not known a priori which ofthe probes in the pair is the perfect match, it is known that when oneprobe specifically hybridizes to a particular target sequence, the otherprobe of the pair will act as a mismatch control for that targetsequence. It will be appreciated that the perfect match and mismatchprobes need not be provided as pairs, but may be provided as largercollections (e.g., 3. 4, 5, or more) of probes that differ from eachother in particular preselected nucleotides. While the mismatch(s) maybe located anywhere in the mismatch probe, terminal mismatches are lessdesirable as a terminal mismatch is less likely to prevent hybridizationof the target sequence. In a particularly preferred embodiment, themismatch is located at or near the center of the probe such that themismatch is most likely to destabilize the duplex with the targetsequence under the test hybridization conditions. In a particularlypreferred embodiment, perfect matches differ from mismatch controls in asingle centrally-located nucleotide.

“Labeled moiety” refers to a moiety capable of being detected by thevarious methods discussed herein or known in the art.

Nucleic Acid Labeling Compounds

The nucleic acid labeling compounds of the present invention are of thefollowing structure:A-O—CH₂-T-H_(c)-L-(M)_(m)-Qwherein A is hydrogen or a functional group that permits the attachmentof the nucleic acid labeling compound to a nucleic acid; T is a templatemoiety; Hc is a heterocyclic group; L is a linker moiety; Q is adetectable moiety; and M is an connecting group, wherein m is an integerranging from 0 to about 5.

The group A is either hydrogen or a functional group that permits theattachment of a nucleic acid labeling compound to a nucleic acid.Nonlimiting examples of such groups include the following:monophosphate; diphosphate; triphosphate (H₄O₉P); phosphoramidite((R₂N)(R′O)P), wherein R is linear, branched or cyclic alkyl, and R′ isa protecting group such as 2-cyanoethyl; and H-phosphonate(HP(O)O—HNR₃), wherein R is linear, branched or cyclic alkyl.

The template moiety (T) is covalently attached to a methylene group(CH₂) at one position and a heterocyclic group (H_(c)) at anotherposition. A nonlimiting set of template moieties is shown in FIG. 1,wherein the substituents are defined as follows: X is O, S, NR₁ or CHR₂;Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; W is O, S orCH₂; D is O or S; and, G is O, NH or CH₂. The substituents R₁, R₂, R₉and R₁₀ are independent of one another and are H, alkyl or aryl.

The heterocyclic group (H_(c)) is a cyclic moiety containing both carbonand a heteroatom. Nonlimiting examples of heterocyclic groupscontemplated by the present invention are shown in FIG. 2.:4-aminopyrazolo[3,4-d]pyrimidine; pyrazolo[3,4-d]pyrimidine; 1,3-diazole(imidazole); 1,2,4-triazine-3-one; 1,2,4-triazine-3,5-dione; and,5-amino-1,2,4-triazine-3-one.

The linker moiety (L) of the nucleic acid labeling compound iscovalently bound to the heterocycle (H_(c)) at one terminal position. Itis attached to the detectable moiety (Q) at another terminal position,either directly or through a connecting group (M). It is of a structurethat is sterically and electronically suitable for incorporation into anucleic acid. Nonlimiting examples of linker moieties include amidoalkyl groups, alkynyl alkyl groups, alkenyl alkyl groups, functionalizedalkyl groups, alkoxyl groups, thio groups and amino alkyl groups.

Amido alkyl groups are of the structure —C(O)NR₃R₄—, wherein R₃ ishydrogen, alkyl or aryl, and R₄ is alkyl or aryl. The amido alkyl groupis preferably of the structure —C(O)NH(CH₂)_(n)R₅—, wherein n is aninteger ranging from about 2 to about 10 and R₅ is O, NR₆ or C(O), andwherein R₆ is hydrogen, alkyl or aryl. More preferably, the amido alkylgroup is of the structure —C(O)NH(CH₂)_(n)N(H)—, wherein n is an integerranging from about 2 to about 6. Most preferably, the amido alkyl groupis of the structure —C(O)NH(CH₂)₄N(H)—.

Alkynyl alkyl groups are of the structure —C≡C—R₄—, wherein R₄ is alkylor aryl. The alkynyl alkyl group is preferably of the structure—C≡C(CH₂)_(n)R₅—, wherein n is an integer ranging from 1 to about 10 andR₅ is O, NR₆ or C(O), and wherein R₆ is hydrogen, alkyl or aryl. Morepreferably, the alkynyl alkyl group is of the structure—C≡C—(CH₂)_(n)N(H)—, wherein n is an integer ranging from 1 to about 4.Most preferably, the alkynyl alkyl group is of the structure—C≡C—CH₂N(H)—.

Alkenyl alkyl groups are of the structure —CH═CH—R₄—, wherein R₄ isalkyl or aryl. The alkenyl alkyl group is preferably of the structure—CH═CH(CH₂)_(n)R₅—, wherein n is an integer ranging from 1 to about 10,and R₅ is O, NR₆ or C(O), and wherein R₆ is hydrogen, alkyl or aryl.More preferably, the alkenyl alkyl group is of the structure—CH═CH(CH₂)_(n)NH—, wherein n is an integer ranging from 1 to about 4.Most preferably, the alkenyl alkyl group is of the structure—CH═CHCH₂NH—.

Functionalized alkyl groups are of the structure —(CH₂)_(n)R₇—, whereinn is an integer ranging from 1 to about 10, and R₇ is O, S, NH, or C(O).The functionalized alkyl group is preferably of the structure—(CH₂)_(n)C(O)—, wherein n is an integer ranging from 1 to about 4. Morepreferably, the functionalized alkyl group is —CH₂C(O)—.

Alkoxy groups are of the structure —O(CH₂)_(n)R₈—, wherein n is aninteger ranging from 2 to about 10, and R₈ is O, S, NH, or C(O). Thealkoxy group is preferably of the structure —O(CH₂)_(n)C(O)—, wherein nis an integer ranging from 2 to about 4. More preferably, the alkoxygroup is of the structure —OCH₂CH₂C(O)—.

Thio groups are of the structure —S(CH₂)_(n)R₈—, wherein n is an integerranging from 2 to about 10, and R₈ is O, S, NH, or C(O). The thio groupis preferably of the structure —S(CH₂)_(n)C(O)—, wherein n is an integerranging from 2 to about 4. More preferably, the thio group is of thestructure —SCH₂CH₂C(O)—.

Amino alkyl groups comprise an amino group attached to an alkyl group.Preferably, amino alkyl groups are of the structure —NH(CH₂)_(n)NH—,wherein n is an integer ranging from about 2 to about 10. The aminoalkyl group is more preferably of the structure —NH(CH₂)_(n)NH—, whereinn is an integer ranging from about 2 to about 4. Most preferably, theamino alkyl group is of the structure —NH(CH₂)₄NH—.

The detectable moiety (Q) is a chemical group that provides an signal.The signal is detectable by any suitable means, including spectroscopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. In certain cases, the signal is detectable by 2 or moremeans.

The detectable moiety provides the signal either directly or indirectly.A direct signal is produced where the labeling group spontaneously emitsa signal, or generates a signal upon the introduction of a suitablestimulus. Radiolabels, such as ³H, ¹²⁵I, ³⁵S, ¹⁴C or ³²P, and magneticparticles, such as Dynabeads™, are nonlimiting examples of groups thatdirectly and spontaneously provide a signal. Labeling groups thatdirectly provide a signal in the presence of a stimulus include thefollowing nonlimiting examples: colloidal gold (40-80 nm diameter),which scatters green light with high efficiency; fluorescent labels,such as fluorescein, texas red, rhodamine, and green fluorescent protein(Molecular Probes, Eugene, Oreg.), which absorb and subsequently emitlight; chemiluminescent or bioluminescent labels, such as luminol,lophine, acridine salts and luciferins, which are electronically excitedas the result of a chemical or biological reaction and subsequently emitlight; spin labels, such as vanadium, copper, iron, manganese andnitroxide free radicals, which are detected by electron spin resonance(ESR) spectroscopy; dyes, such as quinoline dyes, triarylmethane dyesand acridine dyes, which absorb specific wavelengths of light; andcolored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.)beads. See U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241.

A detectable moiety provides an indirect signal where it interacts witha second compound that spontaneously emits a signal, or generates asignal upon the introduction of a suitable stimulus. Biotin, forexample, produces a signal by forming a conjugate with streptavidin,which is then detected. See Hybridization With Nucleic Acid Probes. InLaboratory Techniques in Biochemistry and Molecular Biology; Tijssen,P., Ed.; Elsevier: New York, 1993; Vol.24. An enzyme, such ashorseradish peroxidase or alkaline phosphatase, that is attached to anantibody in a label-antibody-antibody as in an ELISA assay, alsoproduces an indirect signal.

A preferred detectable moiety is a fluorescent group. Flourescent groupstypically produce a high signal to noise ratio, thereby providingincreased resolution and sensitivity in a detection procedure.Preferably, the fluorescent group absorbs light with a wavelength aboveabout 300 nm, more preferably above about 350 nm, and most preferablyabove about 400 nm. The wavelength of the light emitted by thefluorescent group is preferably above about 310 nm, more preferablyabove about 360 nm, and most preferably above about 410 nm.

The fluorescent detectable moiety is selected from a variety ofstructural classes, including the following nonlimiting examples: 1- and2-aminonaphthalene, p,p′diaminostilbenes, pyrenes, quaternaryphenanthridine salts, 9-aminoacridines, p,p′-diaminobenzophenone imines,anthracenes, oxacarbocyanine, marocyanine, 3-aminoequilenin, perylene,bisbenzoxazole, bis-p-oxazolyl benzene, 1,2-benzophenazin, retinol,bis-3-aminopridinium salts, hellebrigenin, tetracycline, sterophenol,benzimidazolyl phenylamine, 2-oxo-3-chromen, indole, xanthen,7-hydroxycoumarin, phenoxazine, salicylate, strophanthidin, porphyrins,triarylmethanes, flavin, xanthene dyes (e.g., fluorescein and rhodaminedyes); cyanine dyes; 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene dyes andfluorescent proteins (e.g., green fluorescent protein,phycobiliprotein).

A number of fluorescent compounds are suitable for incorporation intothe present invention. Nonlimiting examples of such compounds includethe following: dansyl chloride; fluoresceins, such as3,6-dihydroxy-9-phenylxanthhydrol; rhodamineisothiocyanate;N-phenyl-1-amino-8-sulfonatonaphthalene;N-phenyl-2-amino-6-sulfonatonaphthanlene;4-acetamido-4-isothiocyanatostilbene-2,2′-disulfonic acid;pyrene-3-sulfonic acid; 2-toluidinonapththalene-6-sulfonate; N-phenyl,N-methyl 2-aminonaphthalene-6-sulfonate; ethidium bromide; stebrine;auromine-0,2-(9′-anthroyl)palmitate; dansyl phosphatidylethanolamin;N,N′-dioctadecyl oxacarbocycanine; N,N′-dihexyl oxacarbocyanine;merocyanine, 4-(3′-pyrenyl)butryate; d-3-aminodesoxy-equilenin;12-(9′-anthroyl)stearate; 2-methylanthracene; 9-vinylanthracene;2,2′-(vinylene-p-phenylene)bisbenzoxazole; p-bis[2-(4-methyl-5-phenyloxazolyl)]benzene; 6-dimethylamino-1,2-benzophenzin; retinol;bis(3′-aminopyridinium)-1,10-decandiyl diiodide; sulfonaphthylhydrazoneof hellibrienin; chlorotetracycline;N-(7-dimethylamino-4-methyl-2-oxo-3-chromenyl)maleimide;N-[p-(2-benzimidazolyl)phenyl]maleimide; N-(4-fluoranthyl)maleimide;bis(homovanillic acid); resazarin;4-chloro-7-nitro-2,1,3-benzooxadizole; merocyanine 540; resorufin; rosebengal and 2,4-diphenyl-3(2H)-furanone. Preferably, the fluorescentdetectable moiety is a fluorescein or rhodamine dye.

Another preferred detectable moiety is colloidal gold. The colloidalgold particle is typically 40 to 80 nm in diameter. The colloidal goldmay be attached to a labeling compound in a variety of ways. In oneembodiment, the linker moiety of the nucleic acid labeling compoundterminates in a thiol group (—SH), and the thiol group is directly boundto colloidal gold through a dative bond. See Mirkin et al. Nature 1996,382, 607-609. In another embodiment, it is attached indirectly, forinstance through the interaction between colloidal gold conjugates ofantibiotin and a biotinylated labeling compound. The detection of thegold labeled compound may be enhanced through the use of a silverenhancement method. See Danscher et al. J. Histotech 1993, 16, 201-207.

The connecting groups (M)_(m) may serve to covalently attach the linkergroup (L) to the detectable moiety (Q). Each M group can be the same ordifferent and can independently be any suitable structure that will notinterfere with the function of the labeling compound. Nonlimitingexamples of M groups include the following: amino alkyl, —CO(CH₂)₅NH—,—CO—, —CO(O)—, —CO(NH)—, —CO(CH₂)₅NHCO(CH₂)₅NH—, —NH(CH₂CH₂O)_(k)NH—,—NH(CH₂CH₂O)_(k)CH₂CH₂NH and —CO(CH₂)₅—; wherein, k is an integer from 1to about 5, preferably k is 1 or 2; m is an integer ranging from 0 toabout 5, preferably 0 to about 3.

In one embodiment, the nucleic acid labeling compounds of the presentinvention are of the following structure:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; H, is aheterocyclic group; A is H or a functional group that permits theattachment of the nucleic acid label to a nucleic acid; and, M is aconnecting group, wherein m is an integer ranging from 0 to about 3. Thesubstituents R₁, R₂, R₉ and R₁₀ are independent of one another and areH, alkyl or aryl.

In one embodiment, the heterocyclic group (H_(c)) is an imidazole, andthe nucleic acid labeling compounds have the following structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is animidazole and the linking moiety is amido alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; R₃ ishydrogen or alkyl; R₄ is —(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 10; Q is biotin or carboxyfluorescein; A ishydrogen or H₄O₉P₃—; and, M is —CO(CH₂)₅NH— or —CO—, wherein m is 1 or0. More preferably, Y and Z are hydrogen; R₃ is hydrogen; R₄ is—(CH₂)₄NH—; A is H₄O₉P₃—; and, Q is biotin, wherein M is —CO(CH₂)₅NH—and m is 1, or 5- or 6-carboxyfluorescein, wherein m is 0.

In another embodiment, the heterocyclic group (H_(c)) is a C3substituted 4-amino-pyrazolo[3,4-d]pyrimidine, and the nucleic acidlabeling compounds have the following structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is a C3substituted 4-aminopyrazolo[3,4-d]pyrimidine and the linking group is analkynyl alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is aninteger ranging from 1 to about 10; R₅ is O or NH; A is hydrogen orH₄O₉P₃—; Q is biotin or carboxyfluorescein; M is —CO(CH₂)₅NH—, wherein mis 1 or 0. More preferably, Y and Z are OH; n is 1; R₅ is NH; A isH₄O₉P₃—; and, Q is biotin or 5- or 6-carboxyfluorescein, wherein m is 1.

In another embodiment, the heterocyclic group (H_(c)) is an C4substituted pyrazolo[3,4-d]pyrimidine, and the nucleic acid labelingcompounds have the following structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is an N4substituted 4-amino-pyrazolo[3,4-d]pyrimidine and the linking group isan amino alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is aninteger ranging from about 2 to about 10; A is hydrogen or H₄O₉P₃—; Q isbiotin or carboxyfluorescein; M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0. More preferably, Y and Zare hydrogen; n is 4; A is H₄O₉P₃—; Q is biotin or 5- or6-carboxyfluorescein, wherein m is 0.

In another embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3-one, and the nucleic acid labeling compounds have thefollowing structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3-one and the linking group is amino alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is aninteger ranging from about 2 to about 10; A is hydrogen or H₄O₉P₃—; Q isbiotin or carboxyfluorescein; M is —CO(CH₂)₅NH— or—CO(CH₂)₅NHCO(CH₂)₅NH—, wherein m is 1 or 0. More preferably, Y and Zare hydroxyl; n is 4; A is H₄O₉P₃—; Q is biotin or 5- or6-carboxyfluorescein, wherein M is —CO(CH₂)₅NH—, and m is 1.

In another embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3,5-dione, and the nucleic acid labeling compounds havethe following structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is a1,2,4-triazine-3,5-dione and the linking group is alkenyl alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is aninteger ranging from about 1 to about 10; R₅ is NR₆, wherein R₆ ishydrogen, alkyl or aryl; A is hydrogen or H₄O₉P₃—; Q is biotin orcarboxyfluorescein; M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, whereinm is 1 or 0.

In another embodiment, the heterocyclic group (H_(c)) is a5-amino-1,2,4-triazine-3-one, and the nucleic acid labeling compoundshave the following structures:

wherein L is a linker moiety; Q is a detectable moiety; X is O, S, NR₁or CHR₂; Y is H, N₃, F, OR₉, SR₉ or NHR₉; Z is H, N₃, F or OR₁₀; A is Hor a functional group that permits the attachment of the nucleic acidlabel to a nucleic acid; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3. The substituents R₁, R₂, R₉ and R₁₀are independent of one another and are H, alkyl or aryl.

In a preferred embodiment, the heterocyclic group (H_(c)) is a5-amino-1,2,4-triazine-3-one and the linking group is alkenyl alkyl:

wherein Y is hydrogen or hydroxyl; Z is hydrogen or hydroxyl; n is aninteger ranging from about 1 to about 10; R₅ is NR₆, wherein R₆ ishydrogen, alkyl or aryl; A is hydrogen or H₄O₉P₃—; Q is biotin orcarboxyfluorescein; M is —CO(CH₂)₅NH— or —CO(CH₂)₅NHCO(CH₂)₅NH—, whereinm is 1 or 0.

In a preferred embodiment, the nucleic acid labeling compounds have theformulas:

wherein Q is biotin or a carboxyfluorescein.

In another embodiment, the nucleic acid labeling compounds have theformulas:

wherein R₁₁ is hydrogen, hydroxyl, a phosphate linkage, or a phosphategroup; R₁₂ is hydrogen or hydroxyl; R₁₃ is hydrogen, hydroxyl, aphosphate linkage, or a phosphate group; and R₁₄ is a coupled labeledmoiety.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

Stereoisomers are also included within the scope of the invention,examples of two of which are shown below:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is functionalized alkyl; Qis a detectable moiety; and, M is a connecting group, wherein m is aninteger ranging from 0 to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; ; L is —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or a fluorescein; and, a first M is—NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10, and asecond M is —CO(CH₂)_(p)NH—, where p is an interger from about 2 toabout 10, and wherein m is 1 or 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —(CH₂)₂C(O)—, Qis biotin or a carboxyfluorescein; and the first M is —NH(CH₂)₂NH—, andthe second M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is acarboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and a second M is—CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is orbiotin; and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—,wherein m is 2.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+; X is O; Y is OH; Z is OH; L is selected from the group consistingof —CH═CH—C(O)—NH—CH₂—CH₂—NH—C(O)— and —CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O);M is —(CH₂)₅—NH— and Q is biotin having the structure:

In another embodiment, L is selected from the group consisting of—CH═CH—C(O)— Q is a detectable moiety; and, M is a connecting group,wherein m is an integer ranging from 0 to about 3.

In this embodiment, A is preferably selected from the group consistingof H or H₄O₉P₃—; X is preferably O; Y is preferably H or OR₉, wherein R₉is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl oraryl. Q is preferably selected from the group consisting of a biotin ora fluorescein. In this embodiment there is preferably a first M and asecond M wherein the first M is preferably —NH(CH₂)_(n)NH—, wherein n isan integer from about 2 to about 10, the second M is preferably—CO(CH₂)_(p)NH—, wherein p is an interger from about 2 to about 10 and mis 2.

In this embodiment the first M is preferably —NH(CH₂)₂NH— and the secondM is preferably —CO(CH₂)₅NH—.

In another embodiment L comprises a vinyl group (—CH═CH—) attached atits 1 position directly to the N of the base. In accordance with thisaspect of the present invention, where L is vinyl, further substituentsare attached to the 2 position of the vinyl groups such as such as forexample R₁₁(—CH═CH—R₁₁). Preferably R₁₁ is alkyl, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Mostpreferablbly R₁₁ is C(O)R₁₂, where R₁₂ is a bond, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.

In accordance with one aspect of the present invention, R₁₂ ispreferably a bond, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, and Q isselected from the group consisting of a fluorescein and a biotin; and afirst M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, wherein m is 2.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid;

-   -   X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H,        alkyl or aryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H,        alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or        aryl; L is functionalized alkyl; Q is a detectable moiety; and,        M is a connecting group, wherein m is an integer ranging from 0        to about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; ; L is —(CH₂)_(n)C(O)—, wherein n is an integer ranging fromabout 1 to about 10; Q is biotin or a fluorescein; and, a first M is—NH(CH₂)_(n)NH—, wherein n is an integer from about 2 to about 10, and asecond M is —CO(CH₂)₅NH—, wherein m is 1 or 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —(CH₂)₂C(O)—, Qis biotin or a carboxyfluorescein; and a first M is —NH(CH₂)₂NH—, and asecond M is —CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is acarboxyfluorescein; and, a first M is —NH(CH₂)₂NH—, and a second M is—CO(CH₂)₅NH—, wherein m is 2.

In another embodiment, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, Q is orbiotin; and, a first M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—,wherein m is 2.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+; X is O; Y is OH; Z is OH; L is selected from the group consistingof —CH═CH—C(O)—NH—CH₂—CH₂—NH—C(O)— and —CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O);M is —(CH₂)₅—NH— and Q is biotin having the structure:

In another embodiment, L is —CH═CH—C(O)—; Q is a detectable moiety; and,M is a connecting group, wherein m is an integer ranging from 0 to about3.

In this embodiment, A is preferably selected from the group consistingof H or H₄O₉P₃—; X is preferably O; Y is preferably H or OR₉, wherein R₉is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl oraryl. Q is preferably selected from the group consisting of a biotin ora fluorescein. In this embodiment there is preferably a first M and asecond M wherein the first M is preferably —NH(CH₂)_(n)NH—, wherein n isan integer from about 2 to about 10, the second M is preferably—CO(CH₂)_(p)NH—, wherein p is an interger from about 2 to about 10 and mis 2.

In this embodiment the first M is preferably —NH(CH₂)₂NH— and the secondM is preferably —CO(CH₂)₅NH—.

In another embodiment L comprises a vinyl group (—CH═CH—) attached atits 1 position directly to the N of the base. In accordance with thisaspect of the present invention, where L is vinyl, further substituentsare attached to the 2 position of the vinyl groups such as such as forexample R₁₁ (—CH═C—R₁₁). Preferably R₁₁ is alkyl, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl, alkoxy, thio and amino alkyl. Mostpreferablbly R₁₁ is C(O)R₁₂, where R₁₂ is a bond, aryl, functionalizedalkyl, amido alkyl, alkenyl alkyl alkoxy, thio and amino alkyl.Importantly, as disclosed in accordance with one aspect of the presentinvention, the linker group L is selected to provide a linking function,which either alone or in conjunction with appropirate connecting groups(M) appropriately spaces the Q group from the Hc group (nucleotide base)at such a length and in such a configuration as to allow an appropriateassay to be performed on the Q group, but at the same time substantiallypreserving the ability of the nucleic acid labeling compound to act as asubstrate for the appropriate enzyme, e.g., terminal transferase and/orRNA polymerase. Those of skill in the art will also appreciate that thechosen Hc-L-(M)m-Q groups must not adversely impact the ability of anucleic acid strand incorporating such group to undergo Watson-Cricktype base pairing with complementary sequences.

In accordance with one aspect of the present invention, R₁₂ ispreferably a bond, Y is OH; Z is OH; L is —(CH₂)₂C(O)—, and Q isselected from the group consisting of a fluorescein and a biotin; and afirst M is —NH(CH₂)₂NH—, and a second M is —CO(CH₂)₅NH—, wherein m is 2.

In one embodiment, the nucleic acid labeling compounds have thefollowing structures:

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid;

-   -   X is O, S, NR₁ or CHR₂, wherein R₁ and R₂ are, independently, H,        alkyl or aryl; Y is H, N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H,        alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkyl or        aryl; L is amido alkyl; Q is a detectable moiety; and, M is a        connecting group, wherein m is an integer ranging from 0 to        about 3.

In another embodiment, A is H or H₄O₉P₃—; X is O; Y is H or OR₉, whereinR₉ is H, alkyl or aryl; Z is H, N₃, F or OR₁₀, wherein R₁₀ is H, alkylor aryl; ; L is —C(O)NH(CH₂)_(n)NH—, wherein n is an integer rangingfrom about 2 to about 10; Q is biotin or a fluorescein; wherein m is 0,1, or 2.

In another embodiment, Y is H or OH; Z is H or OH; L is —C(O)NH(CH₂)₄NH—or, more preferably, C(O)NH(CH2)₂NH; M is C(O)(CH2)5)NH and Q is biotinor a carboxyfluorescein.

In another embodiment, Y is OH; Z is H; L is —C(O)NH(CH₂)₄NH— or, morepreferably, C(O)NH(CH2)2NH; M is C(O)(CH2)5)NH; Q is biotin.

In another embodiment, Y is OH; Z is H; L is —C(O)NH(CH₂)₄NH— or, morepreferably, C(O)NH(CH2)2NH; M is C(O)(CH2)5)NH; and Q is acarboxyfluorescein.

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+, Y is OH, Z is H or OH, L is —C(O)NH(CH₂)₂NH—, M is —C(O)(CH2)5NH—,n is 1 and Q is biotin, having the structure:

In another embodiment, wherein A is a functional group the permits theattachment of the nucleic acid labeling compound to a nucleic acid;preferably, A is a triphosphate group with apporpriate counterions, saidcounterions selected from the group consisting of H+, Na+, Li+, K+, andNH₄+, Y is OH, Z is H or OH, L is —C(O)NH(CH₂)₂NH—, M is—C(O)((CH₂)₂O)₄(CH₂)₂NH—, n is 1 and Q is biotin, having the structure:

Synthesis of Nucleic Acid Labeling Compounds

FIG. 3 shows a synthetic route to nucleic acid labeling compounds 8a and8b, in which the heterocyclic group (H,) is an imidazole and the linkermoiety (L) is an amido alkyl. The silyl protected imidazole (2) wasadded to pentofuranose (1) to provide a mixture of carboethoxyimidazoledideoxyriboside isomers (3a-3d). The isomers were separated to affordpurified 3c. The carboethoxy group of 3c was converted into an aminocarboxamide (4) upon treatment with a diamine. The terminal amine of 4was protected to give the trifluoroacetylated product 5. The silylprotecting group of 5 was removed, providing the primary alcohol 6.Compound 6 was converted into a 5′-triphosphate to afford 7. Thetrifluoroacetyl protecting group of 7 was removed, and the deprotectedamine was reacted with biotin-NH(CH₂)₅CO—NHS or 5-carboxyfluorescein-NHSgiving, respectively, nucleic acid labeling compounds 8a and 8b.

FIG. 4 shows a synthetic route to C3-labeled4-aminopyrazolo[3,4-d]pyrimidine β-D-ribofuranoside triphosphates. Aprotected propargylamine linker was added to nucleoside (9) underpalladium catalysis to provide the coupled product (10). The primaryalcohol of the alkyne substituted nucleoside (10) was phosphorylated,yielding the 5′-triphosphate 11. The protected amine of triphosphate 11was then deprotected, and the resulting primary amine was treated with areactive biotin or fluorescein derivative to afford, respectively,nucleic acid labeling compounds 12a and 12b.

FIG. 5 shows a synthetic route to pyrazolopyrimidine nucleotides. Achloropyrazolopyrimidine (13) was added to pentofuranose 1 to provideadduct 14 as a mixture of anomers. A diamine was added to compound 14,affording a mixture of primary amines (15). The primary amines (15) wereprotected and chromatographically separated to yield the pure β-anomer16. The silyl group of 16 was removed and the resulting primary alcoholwas phosphorylated to provide triphosphate 17. The trifluoroacetyl groupof 17 was removed and the deprotected amine was treated with a reactivebiotin or carboxyfluorescein derivative giving, respectively, nucleicacid labeling compounds 18a-18d.

FIG. 6 shows a synthetic route to N4-labeled 1,2,4-triazine-3-oneβ-D-ribofuranoside triphosphates. 1,2,4-Triazine-3,5-dioneribonucleoside 19 was converted into the triazole nucleoside 20 upontreatment with triazole and phosphorous trichloride. Addition of adiamine to 20 provided aminoalkyl nucleoside 21. The primary amine of 21was protected, affording trifluoroacetamide 22. The primary alcohol of22 was phosphorylated, and the protected amine was deprotected andreacted with a reactive biotin or carboxyfluorescein derivative, giving,respectively, nucleic acid labeling compounds 23a and 23b.

FIG. 7 shows a synthetic route to C5-labeled 1,2,4-triazine-3,5-dioneriboside phosphates. Aldehyde 24 is reacted with ylide 25 to provide thephthalimide protected allylamine 26. Compound 26 is coupled withpentofuranoside 27, yielding nucleoside 28. The phthalimide group of 28is removed upon treatment with hydrazine to afford primary amine 29.Amine 29 is protected as amide 30. Amide 30 is phosphorylated,deprotected and treated with a reactive derivative of biotin orcarboxyfluorescein, giving, respectively, nucleic acid labelingcompounds 31a and 31b.

FIG. 8 shows a synthetic route to C5-labeled5-amino-1,2,4-triazine-3-one riboside triphosphates. Compound 28 isconverted into the amino-1,3-6-triazine compound 32 upon treatment witha chlorinating agent and ammonia. The phthalimide group of 32 is removedupon treatment with hydrazine, and the resulting primary amine isprotected to provide 33. Compound 33 is phosphorylated, deprotected andtreated with a reactive derivative of biotin or carboxyfluorescein,giving, respectively, nucleic acid labeling compounds 34a and 34b.

Nucleic Acid Labeling

Nucleic acids can be isolated from a biological sample or synthesized,on a solid support or in solution for example, according to methodsknown to those of skill in the art. As used herein, there is notheoretical limitation on the length or source of the nucleic acid usedin the labeling process. Limitations on length may, however, be imposedor suggested by the hybridization process. Exemplary methods of nucleicacid isolation and purification are described in Theory and Nucleic AcidPreparation. In Laboratory Techniques in Biochemistry and MolecularBiology: Hybridization With Nucleic Acid Probes; P. Tijssen, Ed.; PartI; Elsevier: N.Y., 1993. A preferred method of isolation involves anacid guanidinium-phenol-chloroform extraction followed by oligo dTcolumn chromatography or (dT)n magnetic bead use. Sambrook et al.Molecular Cloning: A Laboratory Manual, 2nd ed.; Cold Spring HarborLaboratory, 1989; Vols. 1-3; and Current Protocols in Molecular Biology;F. Ausubel et al. Eds.; Greene Publishing and Wiley Interscience: N.Y.,1987.

In certain cases, the nucleic acids are increased in quantity throughamplification. Suitable amplification methods include, but are notlimited to, the following examples: polymerase chain reaction (PCR)(Innis, et al. PCR Protocols. A guide to Methods and Application;Academic Press: San Diego, 1990); ligase chain reaction (LCR) (Wu andWallace. Genomics 1989, 4, 560; Landgren, et al. Science 1988, 241,1077; and Barringer, et al. Gene 1990, 89, 117); transcriptionamplification (Kwoh et al. Proc. Natl. Acad. Sci. USA 1989, 86, 1173);and self-sustained sequence replication (Guatelli, et al. Proc. Nat.Acad. Sci. USA 1990, 87, 1874). Each of these references is herebyincorporated by reference.

The nucleic acid labeling compound can be incorporated into a nucleicacid using a number of methods. For example, it can be directly attachedto an original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA) or toan amplification product. Methods of attaching a labeling compound to anucleic acid include, without limitation, nick translation,3-end-labeling, ligation, in vitro transcription (IVT) or randompriming. Where the nucleic acid is an RNA, a labeled riboligonucleotideis ligated, for example, using an RNA ligase such as T4 RNA Ligase. SeeThe Enzymes; Uhlenbeck and Greensport, Eds.; Vol. XV, Part B, pp. 31-58;and, Sambrook et al., pp. 5.66-5.69 (incorporated here by reference).Terminal transferase is used to add deoxy-, dideoxy- or ribonucleosidetriphosphates (dNTPs, ddNTPs or NTPs), for example, where the nucleicacid is single stranded DNA.

The labeling compound can also be incorporated at an internal positionof a nucleic acid. For example, PCR in the presence of a labelingcompound provides an internally labeled amplification product. See,e.g., Yu et al. Nucleic Acids Research 1994, 22, 3226-3232 (incorporatedby reference). Similarly, IVT in the presence of a labeling compound canprovide an internally labeled nucleic acid.

Probe Hybridization

The nucleic acid to which the labeling compound is attached can bedetected after hybridization with a nucleic acid probe. Alternatively,the probe can be labeled, depending upon the experimental schemepreferred by the user. The probe is a nucleic acid, or a modifiednucleic acid, that is either attached to a solid support or is insolution. It is complementary in structure to the labeled nucleic acidwith which it hybridizes. The solid support is of any suitable material,including polystyrene based beads and glass chips. In a preferredembodiment, the probe or target nucleic acid is attached to a glasschip, such as a GeneChip® product (Affymetrix, Inc., Santa Clara,Calif.). See International Publication Nos. WO 97/10365, WO 97/29212, WO97/27317, WO 95/11995, WO 90/15070, and U.S. Pat. Nos. 5,744,305 and5,445,934 which are hereby incorporated by reference.

Because probe hybridization is often a step in the detection of anucleic acid, the nucleic acid labeling compound must be of a structurethat does not substantially interfere with that process. The steric andelectronic nature of the labeling compound, therefore, is compatiblewith the binding of the attached nucleic acid to a complementarystructure.

EXAMPLES

The following examples are offered to illustrate, but not to limit, thepresent invention.

General Experimental Details

Reagents were purchased from Aldrich Chemical Company (Milwaukee, Wis.)in the highest available purity. All listed solvents were anhydrous.Intermediates were characterized by ¹H NMR and mass spectrometry.

Example 1 Synthesis of Fluorescein- and Biotin-labeled1-(2,3-dideoxy-β-D-glycero-pentafuranosyl)imidazole-4-carboxamidenucleotides

1-O-acetyl-5-O-(t-butyldimethylsilyl)-2,3-dideoxy-D-glycero-pentafuranose1 (9.4 g, 34.2 mmole) (see, Duelholm, K.; Penderson, E. B., Synthesis,1992, 1) and 1-trimethylsilyl-4-carboethoxyimidazole 2 (6.3 g; 34.2mmole) (see, Pochet, S, et. al., Bioorg. Med. Chem. Lett., 1995, 5,1679) were combined in 100 ml dry DCM under Ar, and trimethylsilyltriflate catalyst (6.2 ml; 34.2 mmole) was added at 0° C. The solutionwas allowed to stir at room temperature for 5 hours and was then washed3× with 100 ml of saturated aqueous NaHCO₃, 1× with saturated aqueousNaCl, dried with NaSO₄ and evaporated to provide 14 g of a crude mixtureof four carboethoxyimidazole dideoxyriboside isomers (3a-d),corresponding to α and β-anomers of both N1 and N3 alkylation products.The isomeric products were purified and separated by flashchromatography (silica gel, EtOAc-hexane), in 52% total yield. The β-N1isomer (2.2 g; 18% yield), was identified by ¹H-NMR chemical shift andNOE data (see, Pochet, S, et. al., Bioorg. Med. Chem. Lett., 1995, 5,1679). Purified 3c (0.5 g; 1.4 mmole) was heated with a 20-fold excessof 1,4-diaminobutane (3.0 ml, 30 mmole) neat at 145° C. for 4 hours, andthen the resulting mixture was diluted with 50 ml EtOAc, washed 3× withwater, 1× with brine, and dried with NaSO₄ and evaporated to provide 500mg (95%) of the imidazole-4-(4-aminobutyl)carboxamide dideoxyriboside 4as a colorless oil. After coevaporation with toluene, 4 (393 mg; 0.75mmole) was combined with trifluoroacetylimidazole (94 uL; 0.83 mmole) in5 ml dry THF at 0° C., and stirred for 10 minutes. The solvent wasevaporated, and the oily residue taken up in 50 ml EtOAc, extracted 2×with saturated aqueous NaHCO₃, 1× with saturated aqueous NaCl, driedwith NaSO₄, and evaporated to yield 475 mg (99%) of the N-TFA protectednucleoside 5 as a colorless oil. The TBDMS group was removed by additionof excess triethylamine trihydrofluoride (2.3 ml; 14.4 mmole) in 20 mldry THF and stirring overnight. The THF was evaporated in vacuo, theresidue was taken up in 50 ml EtOAc and the solution was washedcarefully with a 1:1 mixture of saturated aqueous NaHCO₃ and brine untilneutral, then dried with NaSO₄, and evaporated to yield 340 mg (96%) ofthe 5 as a pale yellow oil. The NMR & MS data were consistent with theassigned structure.

Nucleoside 6 was converted to a 5′-triphosphate, deprotected, reactedwith biotin-NH(CH₂)₅CO—NHS or 5-carboxyfluorescein-NHS and purifiedaccording to procedures reported elsewhere (see, Prober, J. M., et al.,1988, PCT 0 252 683 A2) to give the labeled nucleotides 8a,b in >95%purity by HPLC, ³¹P-NMR.

Example 2 Synthesis of C3-Labeled 4-aminopyrazolo[3,4-d]pyrimidineβ-D-ribofuranoside triphosphates

The synthesis of 3-iodo-4-aminopyrazolo[3,4-d]pyrimidine ribofuranside(9) was carried out as described by H. B. Cottam, et al. 1993, J. Med.Chem. 36:3424. Using the appropriate deoxyfuranoside precursors, boththe 2′-deoxy and 2′,3′-dideoxy nucleosides are prepared using analogousprocedures. See, e.g., U. Neidballa & H. Vorbruggen 1974, J. Org. Chem.39:3654; K. L. Duehom & E. B. Pederson 1992, Synthesis 1992:1).Alternatively, these are prepared by deoxygenation of ribofuranoside 9according to established procedures. See, M. J. Robins et al. 1983 J.Am. Chem. Soc. 103:4059; and, C. K. Chu, et al. 1989 J. Org. Chem.54:2217.

A protected propargylamine linker was added to the4-aminopyrazolo[3,4-d]pyrimidine nucleoside (9) viaorganopalladium-mediated substitution to the 3-position of4-aminopyrazolo[3,4-d]pyrimidine riboside using the procedure describedby Hobbs (J. Org. Chem. 54: 3420; Science 238: 336.). Copper iodide (38mg; 0.2 mmole), triethylamine (560 uL; 4.0 mmole),N-trifluoroacetyl-3-aminopropyne (700 uL; 6.0 mmole) and3-iodo-4-aminopyrazolo[3,4-d]pyrimidine 3-D-ribofuranoside (9) (H. B.Cottam, et al., 1993, J. Med. Chem. 36: 3424.) (786 mg; 2.0 mmole) werecombined in 5 ml of dry DMF under argon. To the stirring mixture wasadded tetrakis(triphenylphosphine) palladium(0) (232 mg; 0.2 mmole). Thesolution became homogeneous within 10 minutes, and was left stirring foran additional 4 hours in the dark, at which time the reaction wasdiluted with 20 mL of MeOH-DCM (1:1), 3.3 g of Dowex AG-1 anion exchangeresin (bicarbonate form) was added, and stirring was continued foranother 15 minutes. The resin was removed by filtration and washed withMeOH-DCM (1:1), and the combined filtrates were evaporated to dryness.The residue was dissolved in 4 mL of hot MeOH, then 15 mL DCM was addedand the mixture kept warm to maintain a homogeneous solution while itwas loaded onto a 5 cm×25 cm column of silica gel that had been packedin 1:9 MeOH-DCM. The product (R_(f)˜0.4, 6:3:1:1 DCM-EtOAc-MeOH-HOAc)was eluted with a 10-15-20% MeOH-DCM step gradient. The resulting paleyellow solid was washed 3× with 2.5 ml of ice-cold acetonitrile, then 2×with ether and dried in vacuo to obtain 630 mg (75%) of4-amino-3-(N-trifluoroacetyl-3-aminopropynyl)pyrazolo[3,4-d]pyrimidineβ-D-ribofuranoside (10). Identity of the product was confirmed by¹H-nmr, mass spectrometry and elemental analysis.

The nucleoside was converted to a 5′-triphosphate (11), deprotected,reacted with oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate, oroxysuccinimidyl-(N-(fluorescein-5-carboxyl)-6-amino)hexanoate, andpurified according to procedures reported elsewhere (Prober, J. M., etal., 1988, PCT 0 252 683 A2.) to give the biotin- andfluorescein-labeled nucleotides (12a, 12b) in >95% purity.

Example 3 Synthesis of Fluorescein- andBiotin-N6-dideoxy-pyrazalo[3,4-d]pyrimidine Nucleotides

1-O-acetyl-5-O(t-butyldimethylsilyl)-2,3-dideoxy-D-glycero-pentofuranose (1) and1-trimethylsilyl-4-chloropyrazolo[3,4-d]pyrimidine (13) were synthesizedaccording to literature procedures. Duelholm, K. L.; Penderson, E. B.,Synthesis 1992, 1-22; and, Robins, R. K., J. Amer Chem Soc. 1995, 78,784-790. To 2.3 g (8.3 mmol) of 1 and 1.9 g (8.3 mmol, 1 eq) of 13 in 40ml of dry DCM at 0° C. under argon was added slowly over 5 minutes 1.5mL (8.3 mmol, 1 eq) of trimethylsilyl triflate. After 30 min. 4.2 ml(41.5 mmol, 5 eq) of 1,2-diaminobutane was added rapidly and thereaction was stirred at room temperature for 1 hr. The solvent wasevaporated; the residue was dissolved in 50 ml of ethylacetate andwashed with 50 ml of saturated aqueous. NaHCO₃ and dried over Na₂SO₄,filtered and the solvent evaporated to yield 4.2 g of a yellow foam. Thefoam was dissolved in 100 ml of diethyl ether and 100 ml of hexanes wasadded to precipitate the product as an oil. The solvent was decanted andthe oil was dried under high vacuum to give 3.4 g of 15 as a pale yellowfoam. HPLC, UV and MS data were consistent with a 2:1 mixture of the α-and β-anomers.

To the crude mixture of isomers (3.4 g, 8.1 mmol, 50% pure) in 140 ml ofdry THF at 0° C. under argon was added slowly 1.0 ml of1-trifluoroacetylimidazole (8.9 mmol, 1.1 eq). The reaction was followedby RP-HPLC. An additional 5% of the acylating agent was added tocompletely convert the starting material to mixture of TFA-protectedanomers. Bergerson, R. G.; McManis, J. S J. Org. Chem 1998, 53,3108-3111. The reaction was warmed to room temperature, and then thesolvent was evaporated to about 25 ml volume and diluted with 100 ml ofethylacetate. The solution was extracted twice with 25 ml of 1% aq.NaHCO₃, once with brine, then dried over Na₂SO₄ and evaporated to afford3.4 g of yellow foam. The crude material was purified by flashchromatography on silica gel in EtOAc-hexanes to give 1.3 g of theα-anomer and 0.7 g of the β-anomer of 16 (50% total yield). The 1H-NMRand MS data were consistent with the assigned structure andstereochemistry.

To 1.3 g (2.5 mmol) of 16 (α-anomer) in 50 ml of dry THF under argon wasadded 1 ml (13.6 mmol) of triethylamine and 6.1 ml (37.5 mmol, 15 eq) oftriethylamine trihydrofluoride. After stirring for 16 hr., the solventwas evaporated, and residual triethylamine trihydrofluoride removedunder high vacuum. Pirrung, M. C.; et al. Biorg. Med. Chem. Lett. 1994,4, 1345-1346. The residue was dissolved in 100 ml of ethylacetate andwashed carefully with 4×100 ml of sat. aq. NaHCO₃, once with brine, thendried over Na₂SO₄ and evaporated to give 850 mg (95%) of white foam.1H-NMR, UV and MS data were consistent with the assigned structure ofthe desilylated nucleoside, which was used in the next step withoutfurther purification.

The nucleoside was converted to the triphosphate using the Ecksteinphosphorylation procedure (Ludwig, J. L. ; Eckstein, F. J. Org. Chem.1989, 54, 631-635) followed by HPLC purification on a ResourceQ anionexchange column (buffer A is 20 mM Tri pH8, 20% CH₃CN and buffer B is 20mM Tris pH8, 1 M NaCl, 20% CH3CN). ³¹P-NMR, UV and MS data wereconsistent with the structure of the triphosphate. Thetrifluoroacetyl-protecting group was removed by treatment with excessNH₄OH at 55° C. for 1 hr. followed by evaporation to dryness. The massspectral data were consistent with the aminobutyl nucleotide 17. Withoutfurther purification, the nucleotide was treated with either Biotin-NHSesters or 5-Carboxyfluorescein-NHS as described elsewhere (Prober, J.M., et al., 1988, PCT 0 252 683 A2) to form the labeled nucleotides18a-18d, respectively, which were purified by HPLC as described (Prober,J. M., et al., 1988, PCT 0 252 683 A2) except that, in the case of 18a,the buffer was 20 mM sodium phosphate pH6. The ³¹P-NMR and UV data wereconsistent with the structure of the labeled analogs.

Example 4 Synthesis of N4-labeled 1,2,4-triazine-3-oneβ-D-ribofuranoside triphosphates

To a solution of 1,2,4-triazole (6.7 g; 97 mmole) in 30 mL dry ACN wasadded POCl₃ (2.1 mL; 22 mmole) slowly with stirring under argon. After30 minutes, the solution was cooled to 0° C., and a solution oftriethylamine (21 mL; 150 mmole) and 2′,3′,5′-tri-O-acetyl-6-azauridine(19, 4.14 g; 11 mmole (commercially available from Aldrich ChemicalCompany)) in 10 mL ACN was added. After stirring for an additional hourat room temperature, the resulting solution of activated nucleoside wastransferred dropwise to a stirring solution of 1,4-diaminobutane (46 g;524 mmole) in 20 mL MeOH. The solvents were removed in vacuo, and theresidue was taken up in water, neutralized with acetic acid, andevaporated again to dryness. The crude residue was purified bychromatography on silica gel (95:5 MeOH—NH₄OH), followed by preparativereverse-phase HPLC to yield 150 mg (0.45 mmole; 3%) of the aminobutylnucleoside (21). This was converted directly to the TFA-protectednucleoside (22) by reaction with 1-trifluoroacetylimidazole (300 uL; 1.8mmole) in 3ml ACN at 0° C. for 2 hours, evaporating the solvent andpurifying by flash chromatography (1:9 MeOH-DCM). Yield 175 mg (0.42mmole; 93%). Identity of the product was confirmed by ¹H-nmr and massspectrometry.

The nucleoside was converted to a 5′-triphosphate, deprotected, reactedwith oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate, oroxysuccinimidyl-(N-(fluorescein-5-carboxyl)-6-amino)hexanoate, andpurified according to procedures reported elsewhere (Prober, J. M., etal., 1988, PCT 0 252 683 A2.) to give the biotin- andfluorescein-labeled nucleotides (23a, 23b) in >95% purity.

Example 5 Synthesis of Biotin and Fluorescein C5-Labeled1,2,4-Triazine-3,5-dione Riboside Triphosphates

5-Formyl-6-azauracil (24) is prepared according to literatureprocedures. See, Scopes, D. I. C. 1986, J. Chem. Med., 29, 809-816, andreferences cited therein. Compound 24 is reacted with the phosphoniumylide of 25, which is formed by treating 25 with catalytic t-butoxide,to provide the phthalimidoyl-protected allylamine 26. Protectedallylamine 26 is ribosylated to provide β-anomer 28 upon reaction of 26with β-D-pentofuranoside 27 (commercially available from Aldrich)according to the procedure of Scopes et al. 1986, J. Chem. Med., 29,809-816. β-ribonucleoside 28 is deprotected with anhydrous hydrazine inTHF to provide allylamine 29. Reaction of primary amine 29 withtrifluoroacetylimidazole in THF affords the protected amine 30.

Nucleoside 30 is converted to a 5′-triphosphate, deprotected, reactedwith oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoate oroxysuccinimidyl-(N-(fluorescein-5-carboxy)-6-amino)hexanoate andpurified according to procedures reported elsewhere (Prober, J. M., etal. 1988, PCT 0 252 683 A2), giving, respectively, the biotin- andfluorescein-labeled nucleotides 31a and 31b.

Example 6 Synthesis of Biotin and Fluorescein C5-Labeled5-Amino-1,2,4-triazine-3-one Riboside Triphosphates

β-ribonucleoside 28, described above, is treated with SOCl₂ or POCl₃ andsubsequently reacted with ammonia to provide the 4-amino-1,3,6-triazinenucleoside 32. The phthalimide group of 32 is removed upon reaction withhydrazine, and the resulting primary amine is protected to affordnucleoside 33. Nucleoside 33 is converted to a 5′-triphosphate,deprotected, reacted with oxysuccinimidyl-(N-biotinoyl-6-amino)hexanoateor oxysuccinimidyl-(N-(fluorescein-5-carboxy)-6-amino)hexanoate andpurified according to procedures reported elsewhere (Prober, J. M., etal. 1988, PCT 0 252 683 A2), giving, respectively, the biotin- andfluorescein-labeled nucleotides 34a and 34b.

Example 7 Procedure for HPLC Analysis of Enzymatic Incorporation ofModified Nucleotides

Reaction Conditions

TdT

-   3 uM dT₁₆ template-   15(30) uM NTP-   40 U TdT (Promega)-   1× buffer, pH 7.5 (Promega)

Procedure: incubate 1 hr. at 37° C., then for 10 min. at 70° C.,followed by the addition of EDTA (2 mM final concentration) in a volumeof 50 uL.

HPLC Analysis

Materials and Reagents

-   4.6 mm×250 mm Nucleopac PA-100 ion-exchange column (Dionex)-   buffer A: 20 mM NaOH (or 20 mM Tris pH 8, in the case of TdT    incorporation of nucleotide triphoshates that are not dye-labeled)-   buffer B: 20 mM NaOH, 1M NaCl (or 20 mM Tris pH 8, 1M NaCl, in the    case of TdT incorporation of nucleotide triphoshates that are not    dye-labeled)    General Procedure

Dilute the reaction with 50 uL of buffer A. Inject 50 uL of this sampleonto the HPLC column and fractionate using a gradient of 5 to 100%buffer B over 30 minutes at a flow rate of 1 mL/min. Detect the peakssimultaneously at 260 nm absorbance and the absorbance maximum of thedye (or the fluorescence emission maximum of the dye).

The incorporation efficiency is expressed as the fraction ofoligonucleotide that is labeled. This number is determined by dividingthe peak area measured at 260 nm absorbance of the labeledoligonucleotide by the sum of the peak areas of the unlabeled andlabeled oligonucleotide. ( The retention time of fluorescein-labeleddT₁₆ is on the order of 2 to 3 min. longer than the unlabeled dT_(16.))The error in this type of assay is about 10%. The percentage labelingefficiency for 4 types of nucleic acid labeling compounds is shown belowin Tables 1, 2 and 3. TABLE 1 Labeling Efficiency

% Labeling Efficiency [TdT]= 160 R B X 40 U U H

—C(O)(CH₂)₅NH—Biotin 100 — H

5-carboxy- fluorescein 94 97 H

5-carboxy- fluorescein 58 98 H

trifluoroacetyl 55 — H

—C(O)(CH₂)₅NH—trifluoroacetyl 49 —

Summary of TdT labeling efficiency data

% Labeling Efficiency [TdT]= X= B= R = linker and label 40 U 160 Ucontrol OH

R = 5-carboxyfluorescein 100 100 control H

R = -biotin 5-carboxyfluorescein 98 97 90 100 analogs: H

R = -biotin —CO(CH₂)₅NH-biotin —CO(CH₂)₅NHCO(CH₂)₅NH-biotin5-carboxyfluorescein 48 41 57 58 100 96 94 98 OH

R = -biotin 5-carboxyfluorescein 6-carboxyfluorescein 25 53 37 84 97 86OH

R = -biotin 54 94

Summary of TdT labeling efficiency data

% Labeling Efficiency [TdT]= X= B= R = linker and label 40 U 160 U H

R = —CO(CH₂)₅NH-biotin 5-carboxyfluorescein 6-carboxyfluorescein 100 9473  —¹²97 99 H

R = -biotin —CO(CH₂)₅NH-biotin —CO(CH₂)₅NHCO(CH₂)₅NH-biotin5-carboxyfluorescein 48 41 57 58 100 96 94 98 OH

R = -biotin 5-carboxyfluorescein 6-carboxyfluorescein 47 67 50 85 98 93OH

R = —CO(CH₂)₅NH-biotin —CO(CH₂)₅NH-fluorescein 98 61 96 88 H

R = —CO(CH₂)₅NH-biotin 5-carboxyfluoresceln 90 81 98 93

Example 8 Hybridization Studies of Labeled Imidazole Carboxamide (“ITP”)and 4-Aminopyrazolo[3,4-d]pyrimidine (“APPTP) Nucleotides

The performance of the labeled imidazolecarboxamide and4-aminopyrazolo[3,4-d]pyrimidine nucleotides was evaluated in a p53assay using standard GeneChip® product protocols (Affymetrix, Inc.,Santa Clara, Calif.), which are described, for example, in detail in theGeneChip® p53 assay package insert. The sample DNA used in theseexperiments was the plasmid “p53mut248.” The labeled nucleotide analogwas substituted for the usual labeling reagent (Fluorescein-N6-ddATP orBiotin-M-N6-ddATP (wherein M=aminocaproyl), from NEN, part #'s NEL-503and NEL-508, respectively). Labeling reactions were carried out usingboth the standard amount of TdT enzyme specified in the assay protocol(25 U) and with 100 U of enzyme. After labeling, Fluorescein-labeledtargets were hybridized to the arrays and scanned directly. Inexperiments using the biotin-labeled targets, the GeneChip® chips werestained in a post-hybridization step with a phycoerythrin-streptavidinconjugate (PE-SA), prior to scanning, according to described procedures(Science 280:1077-1082 (1998)).

FIG. 9 shows comparisons of the observed hybridization fluorescenceintensities for the 1300 bases called in the “Unit-2” part of the chip.In the lower plot, intensities for the Fluorescein-ddITP (8b) labeledtargets are plotted against those for the standard Fluorescein-N6-ddATPlabeled targets (control), both at 25 U of TdT. The observed slope of˜0.75 indicates that the labeling efficiency of 8b was about 75% of thatof Fluorescein-N6-ddATP under these conditions. In the upper plot, thesame comparison is made, except that 100 U of TdT was used in the 8blabeling reaction. The slope of ˜1.1 indicates equivalent or slightlybetter labeling than the standard Fluorescein-N6-ddATP/25 U controlreaction.

FIG. 10 shows comparisons of the observed hybridization fluorescenceintensities for the 1300 bases called in the “Unit-2” part of the chip.Intensities for the Biotin-(M)₂-ddAPPTP (18c, M=aminocaproyl linker;referred to as Biotin-N4-ddAPPTP in FIG. 10) labeled targets (afterPE-SA staining) are plotted against those for the standardBiotin-M-N6-ddATP labeled targets (control), both at 25 U of TdT. Theobserved slope of ˜0.3 indicates that the labeling efficiency withBiotin-(M)₂-ddAPPTP (18c) was about 30% of that of Biotin-M-N6-ddATPunder these conditions.

FIG. 11 shows comparisons of the observed hybridization fluorescenceintensities for the 1300 bases called in the “Unit-2” part of the chip.In the lower plot, intensities for the Biotin-M-ddITP (8a,M=aminocaproyl; referred to as Bio-ddITP in FIG. 11) labeled targets areplotted against those for the standard Biotin-M-N6-ddATP labeled controltargets, both at 25 U of TdT. The observed slope of ˜0.4 indicates thatthe labeling efficiency with 8a was about 40% of that ofBiotin-M-N6-ddATP under these conditions. In the upper plot, the samecomparison is made, except that 100 U of TdT was used in the 8a labelingreaction. The slope of ˜1.1 indicates equivalent or slightly betterlabeling than the standard Biotin-M-N6-ddATP/25 U control reaction.

FIG. 12 shows a comparison of the overall re-sequencing (base-calling)accuracy, for both strands, obtained using Fluorescein-ddITP labeledtargets at both 25 U and 100 U of TdT, as well as the standardFluorescein-N6-ddATP/25 U TdT labeled “control” targets. FIG. 13 shows asimilar comparison for the targets labeled with biotin-M-ddITP (8a;referred to as Biotin-ddITP in FIG. 13) and biotin-M-N6-ddATP “control,”followed by PE-SA staining. FIG. 14 shows a comparison of re-sequencingaccuracy using Biotin-(M)₂-ddAPPTP/100 U TdT and Biotin-M-N6-ddATP/25 UTdT. These data indicate that labeled imidazolecarboxamide and4-aminopyrazolo[3,4-d]pyrimidine dideoxynucleotide analogs can be usedfor DNA target labeling in hybridization-based assays and giveequivalent performance to the standard labeled-N6-ddATP reagent.

Example 9

The performance of the biotin-labeled imidazolecarboxamide and4-aminopyrazolo[3,4-d]pyrimidine nucleotides (“biotin-M-ITP” (8a) and“biotin-(M)₂-APPTP” (18c)) was evaluated using a single-nucleotidepolymorphism genotyping GeneChip® chip array. Published protocols (D. G.Wang, et al., 1998, Science 280: 1077-82.) were used in theseexperiments, except for the following variations: 1) labeling reactionswere carried out using both the standard amount of TdT enzyme specifiedin the published protocol (15 U), or three-fold (45 U) enzyme; 2)substitution of the labeled nucleotide analog for the standard labelingreagent (Biotin-N6-ddATP, from NEN: P/N NEL-508); 3) the labelednucleotide analog was used at either twice the standard concentrationspecified in the published protocol (25 uM), or at six-fold (75 uM).After labeling, biotin-labeled targets were hybridized to the arrays,stained with a phycoerythrin-streptavidin conjugate (PE-SA), and thearray was scanned and analyzed according to the published procedure.

The data is shown in the Table 4 below. As indicated by the meanintensities of the observed hybridization signal (averaged over theentire array), labeling efficiency with biotin-M-ITP (8a) at 25 uM wasas good as Biotin-N6-ddATPat 12.5 uM, and even higher intensity wasgained by using 8a at 75 uM (entries 1-3; 7,8). Compared with thecontrol, this analog provided equivalent or better assay performance,expressed as the ratio of correct base calls. Somewhat lower mean signalintensities are observed with biotin-(M)₂-APPTP (18c), reflecting thelower incorporation efficiency of this analog, but equivalent assayperformance could still be achieved with this analog, using somewhathigher enzyme and nucleotide concentrations (entries 3-6). TABLE 4Comparison of Polymorphism Chip Data Correct [Nucle- Units Mean BaseEntry Sample Nucleotide otide] TdT Intensity Call Ratio 1 A Biotin-M- 7515 164 0.98 ddIcTP (8a) 2 A Biotin-M- 75 45 235 0.98 ddIcTP (8a) 3 BBiotin-N6- 12.5 15 138 0.95 control M-ddATP (NEL 508) 4 B Biotin-N4- 2515 37 0.88 (M)₂- ddAppTP (18c) 5 B Biotin-N4- 75 15 35 0.92 (M)₂-ddAppTP (18c) 6 B Biotin-N4- 75 45 87 0.95 (M)₂- ddAppTP (18c) 7 BBiotin-M- 25 15 116 0.95 ddIcTP (8a) 8 B Biotin-M- 75 15 149 0.95 ddIcTP(8a)

Example 10

High-density DNA probe arrays are proving to be a valuable tool forhybridization-based genetic analysis. These assays require covalentlabeling of nucleic acid molecules with fluorescent or otherwisedetectable molecules in order to detect hybridization to the arrays. Wehave pursued a program to develop a set of novel nucleotide analogs forenzymatic labeling of nucleic acid targets for a variety of array-basedassays. Our primary goal was to provide new reagents for two particularlabeling procedures: (i.), 3′ end labeling of fragmented, PCR-generatedDNA targets with terminal deoxynucleotidyl transferase (TdT); and (ii.),template-directed internal labeling of in vitro transcription-generatedRNA targets with T7 RNA polymerase (T7).

The general approach taken was to screen various base-substitutednucleotide analogs, using a rapid and quantitative HPLC-based assay, toempirically determine which analogs were efficient substrates for thepolymerase of interest. The analogs selected for this study werenucleotides in which the native heterocyclic base was substituted withthe following: 1-(imidazole-4-carboxamide), 1-(1,3,6-trazine-2,4-dione),5-(1,3-pyrimidine-2,4-dione), 3-(pyrazalo-[4,3-d]pyrimidine),1-(pyrazalo-[3,4-d]pyrimidine) and a simple carboxamide moiety. Labeledversions of promising candidate molecules were then designed andsynthesized for further testing of relative incoproation efficiency andfunctional performance in array-based assays.

It was determined that TdT was generally tolerant of base substitutions,and that ribonucleotides were about as efficiently incorporated as2′-deoxy, and 2′,3′-dideoxynucleotides. In contrast, T7 was relativelyintolerant of heterocyclic base substitutions with the exception of the5-(1,3-pyrimidine-2,4-dione), i.e. the pseudo-uridine analog. Two newreagents, a C4-labeled 1-(2′,3′-didexoy-∃-D-ribofuranosyl) imidazole-4carboxamide 5′-triphophate and an N1-labeled pseudo-uridine5′-triphophate, were found to be excellent substrates for TdT and T7,respectively. These new analogs prove array assay performance equivalentto that obtained using conventional labeling reagents.

Example 11 Synthesis of Fluorescent Triphosphate Labels

To 0.5 Emoles (50 μL of a 10 mM solution) of the amino-derivatizednucleotide triphosphate, 3′amino-3′deoxythymidinetriphosphate (1) or2′-amino-2′-deoxyuridine triphosphate (2), in a 0.5 ml ependorf tube wasadded 25 μL of 11 M aqueous solution of sodium borate, pH 7, 87 μL ofmethanol, and 88 μL (10 μmol, 20 wquiv) of a 100 mM solution of5-carboxyfluorescein-X-NHS ester in methanol. The mixture was vortexedbriefly and allowed to stand at room temperature in the dark for 15hours. The sample was then purified by ion-exchange HPLC to afford thefluoresceinated derivatives Formula 3 or Formula 4, below, in about78-84% yield.

Experiments suggest that these molecules are not substrates for terminaltransferase (TdT). It is believed, however, that these molecules wouldbe sutstrates for a polymerase, such as klenow fragment.

Example 12 Synthesis of as-Triazine-3,5[2H,4H]-diones

The analogs as-triazine-3,5[2H,4H]-dione (“6-aza-pyrimidine”)nucleotides (see, FIG. 23 a) are synthesized by methods similar to thoseused by Petrie, et al., Bioconj. Chem. 2: 441 (1991).

Other useful labeling reagents are sythesized including 5-bromo-U/dUTOor ddUTP. See for example Lopez-Canovas, L. Et al., Arch. Med. Res 25:189-192 (1994); Li, X., et al., Cytometry 20: 172-180 (1995); Boultwood,J. Et al., J. Pathol. 148: 61 ff. (1986); Traincard, et al., Ann.Immunol.1340: 399-405 (1983); and FIGS. 23 a, and 23 b set forth herein.

Details of the synthesis of nucleoside analogs corresponding to all ofthe above structures (in particular those of FIG. 23 b) have beendescribed in the literature Known procedcures can be applied in order toattach a linker to the base. The linker modified nucleosides can then beconverted to a triphosphate amine for final attachment of the dye orhapten which can be carried out using commercially available activatedderivatives.

Other suitable labels include non-ribose ornon-2′-deoxyribose-containing structures some of which are illustratedin FIG. 23 c and sugar-modified nucleotide analogues such as areillustrated in FIG. 23 d.

Using the guidance provided herein, the methods for the synthesis ofreagents and methods (enzymatic or otherwise) of label incorporationuseful in practicing the invention will be apparent to those skilled inthe art. See, for example, Chemistry of Nucleosides and Nucleotides 3,Townsend, L. B. ed., Plenum Press, New York, at chpt. 4, Gordon, S. TheSynthesis and Chemistry of Imidazole and Benzamidizole Nucleosides andNucleotides (1994); Gen Chem. Chemistry of Nucleosides and Nucleotides3, Townsend, L. B. ed., Plenum Press, New York (1994); can be made bymethods simliar to those set forth in Chemistry of Nucleosides andNucleotides 3, Townsend, L. B. ed., Plenum Press, New York, at chpt. 4,Gordon, S. “The Synthesis and Chemistry of Imidazole and BenzamidizoleNucleosides and Nucleotides (1994); Lopez-Canovas, L. Et al., Arch. Med.Res 25: 189-192 (1994); Li, X., et al., Cytometry 20: 172-180 (1995);Boultwood, J. Et al., J. Pathol. 148: 61 ff. (1986); Traincard, et al.,Ann. Immunol.1340: 399-405 (1983).

Example 13 Synthesis of N1-Labeled5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a and42b (FIG. 16)

To 5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 39 (100 mg, 0.41mmol, 1 eq.) in acetonitrile (5 ml) was added 1 M TEAB, pH 9 (5 ml)followed by methyl acrylate (5.5 ml, 61 mmol, 150 eq). The reaction wasstirred at room temperature overnight. The solvents were evaporated, andthe residue was coevaporated with water (3×, 5 ml) yielding 135 mg ofacrylate 40. The acrylate 40 was then treated with neat ethylenediamine(2 ml, excess) and two drops of TEA and heated to 100° C. After 1 hourthe excess EDA was evaporated, yielding 146 mg of the free amine(quantitative). The crude residue was then co-evaporated with pyridine(3×, 5 ml, insoluble), resuspended in a mixture of pyridine and DMF andwas cooled to 0° C. To this mixture was added TFA-imidazole (73.8 mg,1.1 eq.). The reaction was then allowed to warm to room temperature andstirred overnight. An additional 1 eq. of TFA-imidazole was added atthis time and the reaction was stirred an additional 15 minutes. Thesolvent was then evaporated, and the residue was co-evaporated withwater(2×, 5 ml) and dissolved in 5 ml of water. The white precipitatethat formed was removed by filtration. The mother liquor, whichcontained the TFA-protected nucleoside 3, was separated into twoaliquots and purified by reverse phase HPLC. The fractions were thenpooled and evaporated to yield 20% (35 mg) of pure 41, which wasverified by ¹H NMR. Using standard procedures (eg., Prober, et al., EP0252683), compound 41 was converted to the triphosphate, which was thenconjugated to biotin and fluorescein to afford 42a and 42b.

Synthesis of the N1-labeled2-amino-5-(β-D-ribofuranosyl)-4(1H)-pyrimidinone, 55, involvedalkylation at N1 using conditions similar to those described byMuehlegger, et al. (WO 96/28640) for the N1-alkylation ofpyrazalo-[4,3-d]pyrimidines (Scheme 2).

The IVT incorporation efficiency (the number of labeled analogsincorporated per transcript) of theN1-fluorescein-X-5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione5′-triphosphate 42a was measured by HPLC (diode array UV detection at260 nm and 495 nm) in an IVT amplification of a 1.24 kb transcript. SeeU.S. patent application Ser. No. 09/126,645 for additional details ontest methods used. Table 1 summarizes the data obtained using differentratios of UTP/5 At a ratio of 1:5, the incorporation and relative yield(measured relative to the yield obtained with UTP only) of transcriptare optimal. This transcript was compared in a hybridization assay totranscript labeled using fluorescein. The preliminary results indicatedthat theN1-fluorescein-X-5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione5′-triphosphate (42a) performed equivalently in a hybridization assay interms of number of correct calls and in hybridization intensity (Charts2 and 3). The hybridization assay used for this purpose was theAffymetrix HIV-PRT GeneChip assay (see Kozal, et al. Nature Medicine1996, 2: 753-9.).

Similarly, the efficiency of DNA 3′-end labeling of a polythymidylateoligonucleotide (T₁₆) using terminal deoxynucleotidyl transferase andN1-fluorescein and biotin-labeled5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate, wasdetermined by HPLC. In this analysis, the percent conversion ofoligo-T₁₆ to the 3′-end labeled T₁₆-Fl, is determined by AX-HPLC (seeU.S. patent application Ser. No. 09/126,645 for detailed procedures).The data is summarized in Chart 4. The incorporation of the biotin andfluorescein triphosphates was very efficient as determined by HPLC.

Chart 1. Incorporation Efficiency of N1-fluorescein-Labeled5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a,determined by HPLC

The labeling reaction conditions are the standard conditions used in theAffymetrix HIV-PRT GeneChip product assay (see Kozal, et al. NatureMedicine 1996, 2: 753-9.).

Chart 2. Call Accuracy of N1-fluorescein-Labeled5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a.

Data was obtained from Affymetrix HIV-PRT GeneChip hybridization assay(see Kozal, et al. Nature Medicine 1996, 2: 753-9.).

Chart 3. Hybridization Signal of fluorescein Labeled triphosphate 42a

Data obtained from hybridization of labeled transcript to the AffymetrixHIV-PRT GeneChip array (see Kozal, et al. Nature Medicine 1996, 2:753-9.).

Chart 4. TdT Labeling Efficiency of Fluorescein and Biotin Labeled5-(β-D-ribofuranosyl)-2,4(1H,3H)-pyrimidinedione 5′-triphosphate 42a and42b, Determined by HPLC

Reaction conditions: TdT (40 units), 20 uM U*TP and 3.2 uM T₁₆ oligo in50 ul of water. Heated at 37° C. for 1 hour and 70° C. for 10 min.,followed by 1 ul of 100 mM EDTA. HPLC analysis was performed on a DionexDNAPac™ PA-100 column.

Example 14 Synthesis of Fluorescein Derivatives of2′-amino-2′-deoxyuridine triphosphate and3′-amino-3′-deoxythymidinetriphosphate (Scheme 3)

To 0.5 umoles (50 uL of a 10 mM solution) of the amino nucleotidetriphosphate (1 or 2) in a 0.5 ml ependorf tube was added 25 ul of a 1 Maqueous solution of sodium borate, pH 8, 87 uL of methanol, and 88 uL(10 umol, 20 equiv) of a 100 mM solution of 5-carboxyfluorescein-X—NHSester in methanol. The mixture was vortexed briefly and allowed to standat room temperature in the dark for 15 hours. The sample was thenpurified by ion-exchange HPLC to afford the fluoresceinated derivatives3 or 4 in about 78-84% yield. Relative efficiencies of incorporation ofthese compounds by TdT are shown in Table 5. TABLE 5 Incorporation oftriphosphate compounds by TdT.

TdT Labeling Efficiencies % Labeled X (3′) Y (2′) B (1′b) 40 U 160 U OHH Uracil 100.0 100.0 NH2 H thymine 100 100 NHCO(CH2)5NH—(CO—FL) Hthymine 1.3 2.2 OH NH2 Uracil 65 95 OH NHCO(CH2)5NH—(CO—FL) Uracil 3.06.6 OH O(CH2)6NH—(CO—FL) Uracil 2.5 7.0 OH O(CH2)6NHCO—(CH2)5-NHCO—Uracil 15.0 17.0 Biotin OH NH(CH2)5CH3 Uracil 4.5 5.0 OH HNHCO(CH2)5NH—(CO— 45.0 55.0 FL)

Example 15 Synthesis ofN-fluorescein-5-carboxamido)ethyl-3-deoxy-allonamide-6-O-triphosphate(FIG. 17)

N1-(di-O-acetylfluorescein-5-carboxamido)ethyl-3-deoxy-4-O-acetyl-6-O-dimethoxytritylallonamide 43 (U.S. patent application Ser. No. 08/574,461) wasdetritylated with 80% acetic acid, and the crude product was purified ona small silica gel column to obtainN1-(di-O-acetylfluorescein-5-carboxamido)ethyl-3-deoxy-4-O-acetylallonamide 44. The allonamide was phosphorylated using POCl₃ followed byreaction with pyrophosphate (Bogachev, Russ. J. Bioorg. Chem. 1996, 22:559-604). The crude product was treated with NH₄OH to remove the acetylprotecting groups, then purified using a preparative Source QTM AX-HPLCcolumn. Pure fractions (analysed by analytical ion-exchange HPLC) werepooled and evaporated to near-dryness. The triphosphate salt 45a wasprecipitated with MeOH-acetone and dried under high vacuum to obtain aproduct which was 98% pure by ion-exchange HPLC and 31p NMR.

Example 16 Synthesis ofN-(6-(fluorescein-5-carboxamido)hexanoyl)-morpholino uridinetriphosphate (Scheme 5)

Morpholino-uracil tosylate salt 1 (30 mg) was co-evaporated withpyridine (3×3 ml) and dissolved in 2 ml of pyridine and cooled to 0° C.Trifluoroacetic anhydride (30 uL) was added and stirred for 1 hour. Thereaction was followed by HPLC until complete. The pyridine was removedand the residue was dissolved in 1 ml of water and filtered. The productwas purified by HPLC on a Waters C-18 bondapak cartridge (Buffer: A=50mM TEM pH 7.0; B=acetonitrile) using a gradient of 0-25%B in 30 minutes(retention time=22 min.). The product was desalted on a Sep-Pakcartridge and freeze-dried to give 151 mg of 2. Phosphorylation of 2using the POCl₃ method gave 3. The removal of the trifluoroacetyl groupwith conc. NH₄OH at 50° C. for 30 min to 4, followed by conjugation to5-carboxyfluoroscein-aminocaproic acid N— hydroxysucciimide (Fl-X-NHS)under standard conditions gave the amide 4. The mass spectral and NMRdata for compounds 1-5 were consistent with the proposed structures.

Example 17 Labeled N-(2-hydroxyethoxy)ethyl 2-O-triphosphates (Scheme6). Example 18 Labeled 2-(2-hydroxyethyl)acetamide 2-O-triphosphates(Scheme 7)

Each of these references is herein incorporated by reference.

Example 19 Synthesis of N-alkyl 2′-amino-2′-deoxyuridine triphosphate(Scheme 8)

Example 20 Synthesis of 2′-O-(6-(Fluorescein-5-carboxamido)hexyl)uridine5′-O-triphosphate (Scheme 9)

Example 21 Synthesis of2′-S(N-(6-(Fluoroescein-5-carboxamido)hexyl)aminoethyldithiouridine5′-O-triphosphate (Scheme 8)

Example 30 Synthesis of Biotin-ΨisoCTP, propenamide-linked (RLR-3b)(Scheme 30)

Peracetylated Pseudoisocytidine 2

Pseudoisocytidine (1) (2.5 g, 9 mmoles) was dissolved in 40 ml drypyridine. Acetic anhydride (8.5 ml, 90 mmoles) was added and the mixturewas stirred under argon for at least 4 hours at room temperature. Thereaction can be monitored by HPLC (C18 column, buffer A: 0.1M TEAA, pH7.5; buffer B: acetonitrile; gradient: 5-95%B over 20 minutes). Thepyridine was removed under vacuum and the residual oil was dissolved in500 ml of ethyl acetate. More ethyl acetate may be added to get a clearsolution since the product has limited solubility in ethyl acetate. Theorganic phase was washed three times with brine and dried over anhydrousNa₂SO₄, filtered and the solvent removed. The white solid wasrecrystallized from ethyl acetate/hexane yielding 3.2 g (85%) of 2.

Propenoic Acid Methyl Ester 3

Compound 2 (2.0 g, 4.86 mmoles) and dimethylaminopyridine (1.2 g, 9.73mmoles) were co-evaporated with 50 ml dry acetonitrile two times andthen re-dissolved in 45 ml dry acetonitrile under argon. Methylpropiolate (0.82 g, 0.86 ml, 9.73 mmoles) was added and the solution wasstirred at room temperature for 24 hours. The reaction turned from acolorless to amber colored solution. The reaction was followed by HPLCuntil no more product was produced. The solvent was removed byrotary-evaporation and the residue was dissolved in 400 ml of ethylacetate and 200 ml of brine. The aqueous layer was back extracted withtwo 200 ml-portions of ethyl acetate. The combined organic layer wasdried over anhydrous Na₂SO₄, filtered and the solvent removed. Theresidue was purified by flash column chromatography on silica gel (200ml wet gel) using ethyl acetate as the eluent affording 850 mg (35%) of3 as a white foam.

Propenoic Acid 4

Compound 3 (0.85 g, 1.7 mmoles) was dissolved in chloroform (5ml) andaqueous concentrated hydrochloric acid (conc., 10 ml) was added. Therosy red solution turned a lemon yellow instantly. The reaction wasstirred at room temperature for an additional 48 hours or until thereaction was complete as determined by RP-HPLC (C18 column, buffer A:0.1M TEAA, pH 7.5; B, acetonitrile; gradient: 0% B for 9 minutes, 0 to90% B over 10 minutes). The solvent and water were removed byrotary-evaporation. The product was purified by precipitation frommethanol/acetonitrile and dried under vacuum to afford 500 mg (94%) of4.

Aminopropenamide 5

Compound 4 (500 mg, 1.6 mmoles) and a buffered solution ofethylenediamine in water (8 ml of 2.0 M ethylenediamine in MES buffer,pH 5.5, containing 16 mmoles of EDA) were mixed and then1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (2 g, 16 mmoles) was addedto the reaction with vigorous stirring. After 1 hour the reaction wasanalyzed by LC/MS and determined to be complete. The compound waspurified by preparative HPLC: PRP-1, 30×250 mm column; flow rate 25ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile; gradient: 0% Bfor 9 minutes, 0 to 90% B over 10 minutes. Salts were removed with aretention time of about 4 min. and the compound eluted from 6 to 7.5minutes. The collected fractions were pooled and the solvent removedunder vacuum. The residue which contained triethylammonium acetate wasco-evaporated with water several times and finally the product wasprecipitated from methanol/acetonitrile to afford 290 mg (51%) of 5.

Biotin-Propenamide 6

Compound 5 (280 mg, 0.79 mmoles) was dissolved in dry DMF (5 ml)followed by the addition of triethylamine (160 mg, 220 μl, 1.58 mmoles).The pH of the solution was adjusted to 7.5 with the addition of moretriethylamine, if necessary. Biotin-X-NHS ester (358 mg, 0.79 mmoles,)was then added to the mixture with stirring. After 1.5 hours the solventvolume was reduced under vacuum to about 1 ml. Caution: do no vacuum todryness because this compound tends to aggregate and it will bedifficult to redissolve. The compound was purified by preparative HPLC:PRP-1, 30×250 mm column; flow rate 25 mmin; buffers: A, 0.1M TEAA, pH7.5, B, acetonitrile; gradient: 0% B for 8 minutes, then 0 to 95% B over20 minutes. Fractions were collected across the peak from 16-17 min andthe solution of pooled fractions was quantitated for the presence ofproduct spectrophotometrically (λ₂₈₉, assuming ε=8000). The solvent wasremoved under vacuum and the residue was co-evaporated with water (30ml) three times and methanol (50 ml) two times. The product wasprecipitated from methanol/acetonitrile yielding 379 mg (69%) of 6.

Triphosphate

Compound 6 (110 mg, 0.1585 mmoles) was dried over P₂O₅ under vacuum fortwo days and then dissolved in trimethyl phosphate (dried over molecularsieves, 20 ml) with gentle heating to about 60° C. Once the materialdissolved, the solution was cooled to ambient temperature and atrap-pack (ABI Trap-pak, P#GEN 084034) was added and the mixture wasallowed to gently stir overnight. The trap-pack was removed and to thesolution at 0° C. under argon was added POCl₃ (73 mg, 45 μl, 0.48mmoles). The reaction was monitored by AX-HPLC for the conversion to themonophosphate, and after 4 hours, an additional 2 equivalents of POCl₃were added and the reaction was allowed to stir for 2 more hours (oruntil 90% conversion was achieved). While monitoring the reaction, asolution of dry tetra(tri-n-butylammonium)pyrophosphate (2.35 mmoles) in5 ml of dry DMF was prepared as follows: n-butylammonium pyrophosphate(Aldrich, P-8533, 1.1 g, 2.35 mmoles) was dissolved in 5 ml dry DMF. Tothe solution was added tri-n-butylamine ( 218 mg, 280 μl, 1.2 mmoles).The solvent was removed under vacuum and the residue was co-evaporatedthree times with 5 ml of dry DMF. To the ammonium salt in 5 ml of dryDMF was added additional tri-n-butylamine (1.12 ml, 2.35 mmoles)]. Thenthe reaction was added drop wise to the pyrophosphate solution withvigorous stirring. After 5 minutes, triethylammonium bicarbonate (1.0 M,pH 7.5, 20 ml) was added to quench the reaction and the mixture was thenanalyzed by HPLC (70% triphosphate). The solution was then diluted 100times with water and loaded directly on to a DEAE ion-exchange columnand purified using standard procedures.

Example 31 Synthesis of Biotin-ΨUTP, Propenamide-Linked (RLR-2B)

Propenoic Acid Methyl Ester 2

Compound 1 (2.5 g, 10.2 mmoles) and dimethylaminopyridine (1.25 g, 10.2mmoles) were dissolved in 125 ml dry DMF under argon. Methyl propiolate(0.943 g, 1.0 ml, 11.2 mmoles) was added and the solution was stirred atroom temperature for 24 hours. The reaction turned from a colorless toamber colored solution. The reaction was followed by HPLC until no moreproduct was produced. The solvent was removed by roto-evaporation andthe residue was dissolved in 10 ml methanol-acetonitrile (1 :1 volume).It was purified by preparative PRP-1, 30×250 mm column using water asbuffer A and acetonitrile as buffer B with a flow rate 25 ml/min.Eluting from 5 to 95% B in 15 minutes. Collect the fraction from 9 to 10minute. Remove solvent to afford 1.1 g (33%) as a white solid.

Propenoic Acid 3

Compound 2 (1.1 g, 3.35 mmoles) was dissolved in 80 ml 1.0 N HCl andheated to 60° C. for 88 hours when LC-MS indicated the starting materialis completed converted. The reaction mixture was evaporated to an oilyresidual by rotary-evaporation and redissolve in minimum amount ofmethanol. Add the methanol solution slowly to acetonitrile (at least 200ml) to precipitate the free acid. Collect the solid and dried undervacuum to afford 1.0 g (94%) of white solid.

Aminopropenamide 4

Compound 3 (1.0 g, 3.18 mmoles) and a buffered solution ofethylenediamine in water (16 ml of 2.0 M ethylenediamine in 0.1 M MESbuffer, pH 5.5, containing 32 mmoles of EDA) were mixed and then1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4 g, 32 mmoles) was addedto the reaction with vigorous stirring. After 1 hour the reaction wasanalyzed by LC/MS and determined to be complete. Note: desalt a samplefor LC-MS. The compound was purified by preparative HPLC: PRP-1, 30×250mm column; flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B,acetonitrile; gradient: 0% B for 9 minutes, 0 to 90% B over 10 minutes.Salts were removed with a retention time of about 4 min. and thecompound eluted from 6 to 7.5 minutes. The fractions were pooled and thesolvent removed under vacuum. The residue which containedtriethylammonium acetate was co-evaporated with water several times andfinally the product was precipitated from methanol/acetonitrile toafford 700 mg (62%) of 4.

Biotin-Propenamide 5

Compound 4 (102 mg, 0.286 mmoles) was co-evaporated with dry DMF twice(5 ml each) and then dissolved in dry DMF (1.5 ml) followed by theaddition of triethylamine (29 mg, 40 μl, 0.286 mmoles). The pH of thesolution was adjusted to 7.5 with the addition of more triethylamine, ifnecessary. Biotin-X-NHS ester (0.286 mmoles, 130 mg) was then added tothe mixture with stirring. After 1.0 hour, the reaction was monitored byHPLC for completion. The solvent volume was reduced under vacuum toabout 1 ml. Caution: do not vacuum to dryness because this compoundtends to aggregate and it will be difficult to redissolve. The residualwas redissolved in 5 ml water and 1 ml methanol.

The compound was purified by preparative HPLC: PRP-1, 30×250 mm column;flow rate 25 ml/min; buffers: A, 0.1M TEAA, pH 7.5, B, acetonitrile;gradient: 0% B for 11 minutes, then 0 to 95% B over 16 minutes.Fractions were collected across the peak from 19-21 min. The solvent wasremoved under vacuum and the residue was co-evaporated with water (30ml) three times and methanol (50 ml) two times. The product wasrecrystallized from acetonitrile yielding 130 mg (67%) of 5.

Triphosphate 6

Compound 5 (130 mg, 0.187 mmoles) was dried over P₂O₅ under vacuum for24 hours and then dissolved in trimethyl phosphate (dried over molecularsieves, 20 ml) with gentle heating to about 60° C. Once the materialdissolved the solution was cooled to ambient temperature and a trap-pack(ABI Trap-pak, P#GEN 084034) was added and allowed to gently stirovernight. The solution turned into a little cloudy when chilled on ice.The trap-pack was removed and to the solution at 0° C. under argon wasadded POCl₃ (115 mg, 70 μl, 0.748 mmoles). The reaction was monitored byAX-HPLC for the conversion to the monophosphate, and after 4 hours, anadditional one equivalent of POCl₃ were added and the reaction wasallowed to stir for 2 more hours (90% conversion). While monitoring thereaction, a solution of dry tetra(tri-N-butylammonium)pyrophosphate(0.187×5×3.3=3.1 mmoles) in 6 ml dry DMF was prepared. Then the reactionwas added drop wise to the pyrophosphate solution with vigorousstirring. After 5 minutes, triethylammonium bicarbonate (1.0 M, pH 7.5,23 ml) was added to quench the reaction. The mixture was stirred on icefor 30 minutes and placed in a fridge overnight. The mixture was thenanalyzed by HPLC (70% triphosphate) and purified using standard TriLinkprocedures on DEAE.

The final reaction mixture may be diluted with mili Q water by a factorof 100, and then loaded on DEAE column. It is not recommended to rotovapoff TEAB because the compound may be unstable under basic condition.

To prepare tetra(tri-N-butylammonium)pyrophosphate, TBA-PPi (Aldrich,P-8533, 1.5 TBA per PPi, 1.4 g, 3.1 mmoles) was dissolved in 5 ml dryDMF.

Add TBA 287 mg, 364 μl, 1.55 mmoles). Co-evaporate with 5 ml dry DMF atleast three times. Redissolve in 5 ml anhydrous DMF. Add TBA (1.46 ml,3.1 mmoles). Handle the materials in a glove box filled with Ar.

Example 32 (Synthesis of Biotin-Virtual NTP) (Schemes 32)

The Allonic methyl ester 1 (2.5 g,) was treated with 20 mlethylenediamine (as solvent) at 25° C. for 24 hrs. The EDA was removedby rotary evaporation and the residue was co-evaporated several timeswith water until all of the EDA was removed (as measured by TLC onsilica in 10% MeOH/DCM using ninhydrin stain) affording allonamide,quantitatively. The extent of reaction was analyzed by RP-HPLC and thenwas characterized by HNMR/MS. The purity was good with very littlecontaminating EDA present so that the allonamide was taken on to thenext step without further purification. To the allonamide in DMF wereadded 1.1 equivalents of Biotin-X-NHS (commercially available fromClonetech) followed by 2 equivalents of triethylamine. The solution wasstirred overnight at room temperature and analyzed by HPLC for extent ofreaction. The solvent was then removed by evaporation and the residuewas purified by column chromatography on silica eluting with a linear,stepwise gradient of 1-10% MeOH/DCM to afford 50% of 2 (NMR/MS seeattachment). The trityl group of 2 was removed by treatment with 20 mlof a solution of 10% TFA/MeOH. The reaction was monitored by TLC, 10%MeOH/DCM and visualized using an acidic solution ofdimethylaminocinnamanaldehyde/EtO. The reaction was diluted with DCM andthe product was extracted into water. The water was removed by rotaryevaporation and the residue was purified by precipitation by slowaddition of a methanol solution of the biotinylated product to ethylacetate to form 196 mg (71%) of pure biotinylated nucleoside. The yellowsticky foam was co-evaporated with dry pyridine 3-times to remove waterand used directly in the phosphorylation reaction (scheme 32a).

At this point, the nucleoside was phosphorylated to 3 using standardconditions for the preparation of dexyribonucleotide triphosphates².

Example 33

IVT incorporation was determined spectrophotometrically using 260 nmabsorbance for the quantitation of RNA and a HABA-based colorimetricassay for quantitation of biotin for RLR-3b (Biotin-ΨisoCTP,vinyl-linked), RLR-2b (Biotin-ψUTP, vinyl-linked) and RLR-2a(Biotin-ΨUTP, ethane (or saturated)-link). The vinyl linked analogs wereboth incorporated more efficiently than the saturated ethane analog asshown in FIG. 21. Purified and fragmented RNA was quantitated by UVabsorbance at 260 nm, and the amount of biotin incorporated wasdetermined using a spectrophotometric-based assay for biotin (see, e.g.,Swaisgood, H. E. et al 1996 Applied Biochemistry and Biotechnology,56,1.)

Example 34

This example describes a simple and mild, one-step method for theconversion of a pseuodoisocytidine molecule to a pseudouridine molecule.

Pseudoisocytidine carboxylic acid (1) was condensed with one equivalentof (biotin-ε-aminocaprolamido)ethylamine at pH 5.5 in 100 mM bufferusing excess EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride) to form the biotin labeled pseudouridine (2) in 60%yield, after reverse-phase HPLC purification. Apparently the EDCcatalyzes not only amide formation but also activates the C2 exocyclicamino group of the pyrimidine ring for subsequent hydrolysis, thustransforming the pseudoisocytidine to a pseudouridine ring system. Therepresents a mild, one-step method for this kind of transformation,which usually requires more reactive activating agents and results inadditional side reactions.

All patents, patent applications, and literature cited in thespecification are hereby incorporated by reference in their entirety. Inthe case of any inconsistencies, the present disclosure, including anydefinitions therein will prevail.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

1. A method for converting a pseudoisocytidine base to a pseuodouridinebase, said method comprising the steps of providing a pseudoisocytidinemolecule having the formula

wherein A is H or a functional group that permits the attachment of thenucleic acid labeling compound to a nucleic acid; X is O, S, NR₁ orCHR₂, wherein R₁ and R₂ are, independently, H, alkyl or aryl; Y is H,N₃, F, OR₉, SR₉ or NHR₉, wherein R₉ is H, alkyl or aryl; Z is H, N₃, For OR₁₀, wherein R₁₀ is H, alkyl or aryl; L is a linker group selectedfrom the group consisting of —CH═CH—R₁₁, or C≡C—R₁₁ wherein R₁₁comprises a moiety selected from the group consisting of alkyl,functionalized alkyl, alkenyl alkyl, alkynal alkyl, amido alkyl, aminoalkyl, alkoxy, amino, aryl, and thio; Q is a detectable moiety; and, Mis a connecting group, and wherein m is an integer ranging from 0 toabout 3; incubating said pseudoisocytidine molecule with EDC to providea pseudouridine molecule having the formula:

wherein Q, (M)m, L, A, X, Y and Z have the meaning ascribed above.
 2. Amethod according to claim 1 wherein A is selected from the groupconsisting of H, α-thiotriphosphate and H₄O₉P₃—; X is O; Y is H or OH; Zis H or OR₁₀, wherein R₁₀ is H, alkyl or aryl.
 3. A method according toclaim 2 wherein Q is selected from the group consisting of colloidalgold, a fluorescent group and biotin.
 4. A method according to claim 3wherein Q is a biotin.
 5. A method according to claim 3 having a first Mand a second M, wherein said first M is —NH(CH₂)_(n)NH—, wherein n is aninteger from about 2 to about 10, and a second M is —CO(CH₂)_(p)NH—,wherein p is an integer from about 2 to about 10 and m is
 2. 6. A methodaccording to claim 5 wherein said first M is —NH(CH₂)₂NH— and saidsecond M is —CO(CH₂)₅NH—.
 7. A method according to claim 1 wherein A isa triphosphate group having appropriate counterions and Y is OH and Z isOH.
 8. A method according to claim 7 wherein said counterions areselected from the group consisting of H⁺, Na⁺, Li⁺, K⁺, (CH₃CH₂)₃NH⁺ andNH₄ ⁺.
 9. A method for converting a pseudoisocytidine base to abiotinylated pseuodouridine, said method comprising the steps ofproviding a pseudoisocytidine base having the formula

condensing said pseudoisocytidine molecule with one equivalent of(biotin-ε-aminocaprolamido)ethylamine using excess EDC(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to form abiotinylated pseudouridine compound having the formula:


10. A method according to claim 9 wherein said step of condensing iscarried out at pH 5.5 in MES buffer.